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WO2013028928A1 - Cellulolytic enzyme compositions and uses thereof - Google Patents

Cellulolytic enzyme compositions and uses thereof Download PDF

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Publication number
WO2013028928A1
WO2013028928A1 PCT/US2012/052163 US2012052163W WO2013028928A1 WO 2013028928 A1 WO2013028928 A1 WO 2013028928A1 US 2012052163 W US2012052163 W US 2012052163W WO 2013028928 A1 WO2013028928 A1 WO 2013028928A1
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WO
WIPO (PCT)
Prior art keywords
seq
mature polypeptide
beta
sequence
cellobiohydrolase
Prior art date
Application number
PCT/US2012/052163
Other languages
French (fr)
Inventor
Jeffrey Shasky
Suchindra Maiyuran
Amanda Fischer
Original Assignee
Novozymes, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to DK12756598.4T priority Critical patent/DK2748305T3/en
Priority to EP12756598.4A priority patent/EP2748305B1/en
Priority to CA2846398A priority patent/CA2846398A1/en
Priority to US14/238,431 priority patent/US9476036B2/en
Application filed by Novozymes, Inc. filed Critical Novozymes, Inc.
Priority to AU2012298725A priority patent/AU2012298725C1/en
Priority to CN201280052443.4A priority patent/CN103890165A/en
Priority to BR112014004186-5A priority patent/BR112014004186B1/en
Priority to IN2100CHN2014 priority patent/IN2014CN02100A/en
Publication of WO2013028928A1 publication Critical patent/WO2013028928A1/en
Priority to US15/265,460 priority patent/US10081824B2/en
Priority to AU2017219076A priority patent/AU2017219076B2/en
Priority to US16/122,173 priority patent/US10233473B2/en
Priority to AU2019200618A priority patent/AU2019200618B2/en
Priority to US16/268,922 priority patent/US10662456B2/en
Priority to US16/878,267 priority patent/US11230723B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01176—Cellulose 1,4-beta-cellobiosidase (reducing end)(3.2.1.176)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • the present invention relates to cellulolytic enzyme compositions; recombinant filamentous fungal host cells producing the cellulolytic enzyme compositions and methods of producing and using the compositions.
  • Cellulose is a polymer of glucose linked by beta-1 ,4-bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta- glucosidases hydrolyze cellobiose to glucose.
  • lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.
  • WO 201 1/057140 discloses an Aspergillus fumigatus cellobiohydrolase I and gene thereof.
  • WO 201 1/057140 discloses an Aspergillus fumigatus cellobiohydrolase II and gene thereof.
  • WO 2005/047499 discloses an Aspergillus fumigatus beta-glucosidase and gene thereof.
  • WO 2006/078256 discloses Aspergillus fumigatus GH10 xylanases.
  • WO 201 1/057140 discloses an Aspergillus fumigatus beta-xylosidase and gene thereof.
  • WO 201 1/041397 discloses a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity and gene thereof.
  • the present invention provides cellulolytic enzyme compositions and methods of producing and using the compositions.
  • the present invention relates to enzyme compositions, comprising (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the present invention also relates to recombinant filamentous fungal host cells, comprising polynucleotides encoding (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the present invention also relates to methods of producing an enzyme composition, comprising: (a) cultivating a filamentous fungal host cell of the present invention under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
  • the present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition of the present invention.
  • the present invention also relates to processes for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • the present invention further relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition of the present invention.
  • Figure 1 shows a restriction map of plasmid pJfyS139.
  • Figure 2 shows a restriction map of plasmid pJfyS142.
  • Figure 3 shows a restriction map of plasmid pJfyS144.
  • Figure 4 shows a restriction map of plasmid pDM286.
  • Figure 5 shows a restriction map of plasmid pDFng1 13-3.
  • Figure 6 shows a restriction map of plasmid pSMai139.
  • Figure 7 shows a restriction map of plasmid pSMai143.
  • Figure 8 shows a restriction map of plasmid pSMai229.
  • Figure 9 shows a restriction map of plasmid pAG57.
  • Figure 10 shows a restriction map of plasmid pDFng124-1.
  • Figure 1 1 shows a restriction map of plasmid pSaMe-AFGH10.
  • Figure 12 shows a comparison of percent conversion of pretreated corn stover (PCS) by an enzyme composition comprising an Aspergillus fumigatus cellobiohydrolase I; an Aspergillus fumigatus cellobiohydrolase II; an Aspergillus fumigatus beta-glucosidase variant; a Penicillium sp.
  • PCS pretreated corn stover
  • GH61 polypeptide having cellulolytic enhancing activity, an Aspergillus fumigatus xylanase, and an Aspergillus fumigatus beta-xylosidase ("enzyme composition #1 ") to an enzyme composition comprising a blend of an Aspergillus aculeatus GH10 xylanase and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide ("enzyme composition #2").
  • Acetylxylan esterase means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate.
  • acetylxylan esterase activity is determined using 0.5 mM p- nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01 % TWEENTM 20 (polyoxyethylene sorbitan monolaurate).
  • One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • allelic variant means any of two or more (e.g., several) alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • Alpha-L-arabinofuranosidase means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L- arabinosides.
  • the enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans.
  • Alpha-L- arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L- arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase.
  • alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co.
  • Alpha-glucuronidase means an alpha-D- glucosiduronate glucuronohydrolase (EC 3.2.1 .139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol.
  • alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249.
  • One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 ⁇ of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40°C.
  • Aspartic protease means a protease that uses an aspartate residue(s) for catalyzing the hydrolysis of peptide bonds in peptides and proteins. Aspartic proteases are a family of protease enzymes that use an aspartate residue for catalytic hydrolysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH (Szecsi, 1992, Scand. J. Clin. Lab. In vest. Suppl. 210: 5-22). For purposes of the present invention, aspartic protease activity is determined according to the procedure described by Aikawa et al., 2001 , J. Biochem. 129: 791 -794.
  • Beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1 .21 ) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose.
  • beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66.
  • beta-glucosidase is defined as 1 .0 ⁇ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p- nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20 (polyoxyethylene sorbitan monolaurate).
  • Beta-xylosidase means a beta-D-xyloside xylohydrolase (E.C. 3.2.1 .37) that catalyzes the exo-hydrolysis of short beta ⁇ (4) xylooligosaccharides to remove successive D-xylose residues from non-reducing termini.
  • one unit of beta-xylosidase is defined as 1 .0 ⁇ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D- xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • Cellobiohydrolase means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1 .176) that catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.
  • Cellulolytic enzyme or cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • the two basic approaches for measuring cellulolytic activity include: (1 ) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481.
  • Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
  • the most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).
  • cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, compared to a control hydrolysis without addition of cellulolytic enzyme protein.
  • Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnS0 4 , 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMI NEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
  • Cellulosic material means any material containing cellulose.
  • the predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin.
  • the secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose.
  • Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents.
  • cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
  • Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • the cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E.
  • the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.
  • the cellulosic material is any biomass material.
  • the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.
  • the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue).
  • the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switchgrass. In another aspect, the cellulosic material is wheat straw.
  • the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
  • the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.
  • the cellulosic material is an aquatic biomass.
  • aquatic biomass means biomass produced in an aquatic environment by a photosynthesis process.
  • the aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.
  • the cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide.
  • Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • Endoglucanase means an endo-1 ,4-(1 ,3;1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • endoglucanase means an endo-1 ,4-(1 ,3;1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ).
  • endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • Family 61 glycoside hydrolase The term “Family 61 glycoside hydrolase” or “Family GH61 “ or “GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695- 696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member.
  • Feruloyl esterase means a 4-hydroxy-3- methoxycinnamoyl-sugar hydrolase (EC 3.1 .1.73) that catalyzes the hydrolysis of 4-hydroxy- 3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).
  • Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II.
  • feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0.
  • One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • Flanking means DNA sequences extending on either side of a specific DNA sequence, locus, or gene.
  • the flanking DNA is immediately adjacent to another DNA sequence, locus, or gene that is to be integrated into the genome of a filamentous fungal cell.
  • fragment means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide main; wherein the fragment has enzyme activity.
  • a fragment contains at least 85%, e.g., at least 90% or at least 95% of the amino acid residues of the mature polypeptide of an enzyme.
  • Hemicellulolytic enzyme or hemicellulase means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass.
  • hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
  • the substrates of these enzymes are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation.
  • the catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups.
  • GHs glycoside hydrolases
  • CEs carbohydrate esterases
  • catalytic modules based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A).
  • GH-A GH-A
  • a most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.
  • High stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • homologous 3' region means a fragment of DNA that is identical in sequence or has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a region in the genome and when combined with a homologous 5' region can target integration of a piece of DNA to a specific site in the genome by homologous recombination.
  • homologous 5' region means a fragment of DNA that is identical in sequence to a region in the genome and when combined with a homologous 3' region can target integration of a piece of DNA to a specific site in the genome by homologous recombination.
  • the homologous 5' and 3' regions must be linked in the genome which means they are on the same chromosome and within at least 200 kb of one another.
  • homologous flanking region means a fragment of DNA that is identical or has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a region in the genome and is located immediately upstream or downstream of a specific site in the genome into which extracellular DNA is targeted for integration.
  • homologous repeat means a fragment of DNA that is repeated at least twice in the recombinant DNA introduced into a host cell and which can facilitate the loss of the DNA, i.e., selectable marker that is inserted between two homologous repeats, by homologous recombination.
  • a homologous repeat is also known as a direct repeat.
  • Host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • Non-limiting examples of isolated substances include (1 ) any non- naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • Low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide of an A. fumigatus cellobiohydrolase I is amino acids 27 to 532 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6) that predicts amino acids 1 to 26 of SEQ ID NO: 2 are a signal peptide.
  • the mature polypeptide of an A is amino acids 27 to 532 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6) that predicts amino acids 1 to 26 of SEQ ID NO: 2 are a signal peptide.
  • fumigatus cellobiohydrolase II is amino acids 20 to 454 of SEQ ID NO: 4 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 4 are a signal peptide.
  • the mature polypeptide of an A. fumigatus beta-glucosidase is amino acids 20 to 863 of SEQ ID NO: 6 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 6 are a signal peptide.
  • GH61 polypeptide is amino acids 26 to 253 of SEQ ID NO: 8 based on the SignalP program that predicts amino acids 1 to 25 of SEQ ID NO: 8 are a signal peptide.
  • the mature polypeptide of an A. fumigatus xylanase I is amino acids 18 to 364 of SEQ ID NO: 10 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 10 are a signal peptide.
  • the mature polypeptide of an A. fumigatus xylanase II is amino acids 20 to 323 of SEQ ID NO: 12 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 12 are a signal peptide.
  • the mature polypeptide of an A. fumigatus xylanase III is amino acids 20 to 397 of SEQ ID NO: 14 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 14 are a signal peptide.
  • the mature polypeptide of an A. fumigatus beta-xylosidase is amino acids 21 to 792 of SEQ ID NO: 16 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 16 are a signal peptide.
  • the mature polypeptide of a T. reesei cellobiohydrolase I is amino acids 18 to 514 of SEQ ID NO: 18 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 18 are a signal peptide.
  • the mature polypeptide of a T. reesei cellobiohydrolase II is amino acids 19 to 471 of SEQ ID NO: 20 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 20 are a signal peptide.
  • reesei beta-glucosidase is amino acids 20 to 744 of SEQ ID NO: 22 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 22 are a signal peptide.
  • the mature polypeptide of a T. reesei xylanase I is amino acids 20 to 229 of SEQ ID NO: 24 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 24 are a signal peptide.
  • the mature polypeptide of a T. reesei xylanase II is amino acids 20 to 223 of SEQ ID NO: 26 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 26 are a signal peptide.
  • the mature polypeptide of a T. reesei xylanase III is amino acids 17 to 347 of SEQ ID NO: 28 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 28 are a signal peptide.
  • the mature polypeptide of a T is amino acids 20 to 223 of SEQ ID NO: 26 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 26 are a signal peptide.
  • the mature polypeptide of a T. reesei xylanase III is amino acids 17 to 347 of SEQ ID NO: 28 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO:
  • reesei beta-xylosidase is amino acids 21 to 797 of SEQ ID NO: 30 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having enzyme activity.
  • the mature polypeptide coding sequence of an A. fumigatus cellobiohydrolase I is nucleotides 79 to 1596 of SEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP program (Nielsen et al. , 1997, supra) that predicts nucleotides 1 to 78 of SEQ ID NO: 1 encode a signal peptide.
  • the mature polypeptide coding sequence of an A is nucleotides 79 to 1596 of SEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP program (Nielsen et al. , 1997, supra) that predicts nucleotides 1 to 78 of SEQ ID NO: 1 encode a signal peptide.
  • the mature polypeptide coding sequence of an A is nucleotides 79 to 1596 of SEQ ID NO: 1 or the c
  • fumigatus cellobiohydrolase II is nucleotides 58 to 1700 of SEQ ID NO: 3 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 3 encode a signal peptide.
  • the mature polypeptide coding sequence of an A. fumigatus beta-glucosidase is nucleotides 58 to 2580 of SEQ ID NO: 5 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 5 encode a signal peptide.
  • the mature polypeptide coding sequence of a Penicillium sp is nucleotides 58 to 1700 of SEQ ID NO: 3 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 3 encode a signal peptide.
  • GH61 polypeptide is nucleotides 76 to 832 of SEQ ID NO: 7 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 7 encode a signal peptide.
  • the mature polypeptide coding sequence of an A. fumigatus xylanase I is nucleotides 52 to 1 145 of SEQ ID NO: 9 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 9 encode a signal peptide.
  • the mature polypeptide coding sequence of an A is nucleotides 76 to 832 of SEQ ID NO: 7 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 7 encode a signal peptide.
  • the mature polypeptide coding sequence of an A. fumigatus xylanase I is nucleotides 52 to 1
  • fumigatus xylanase II is nucleotides 58 to 1400 of SEQ ID NO: 1 1 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 1 1 encode a signal peptide.
  • the mature polypeptide coding sequence of an A. fumigatus xylanase III is nucleotides 107 to 1415 of SEQ ID NO: 13 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 106 of SEQ ID NO: 13 encode a signal peptide.
  • the mature polypeptide coding sequence of an A is nucleotides 58 to 1400 of SEQ ID NO: 1 1 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 1 1 encode a signal peptide.
  • fumigatus beta-xylosidase is nucleotides 61 to 2373 of SEQ ID NO: 15 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 15 encode a signal peptide.
  • the mature polypeptide coding sequence of a T. reesei cellobiohydrolase I is nucleotides 52 to 1545 of SEQ ID NO: 17 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 17 encode a signal peptide.
  • the mature polypeptide coding sequence of a T. reesei cellobiohydrolase II is nucleotides 55 to 1608 of SEQ ID NO: 19 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 19 encode a signal peptide.
  • the mature polypeptide coding sequence of a T is nucleotides 52 to 1545 of SEQ ID NO: 17 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 17 encode a signal peptide.
  • reesei beta-glucosidase is nucleotides 58 to 2612 of SEQ ID NO: 21 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 21 encode a signal peptide.
  • the mature polypeptide coding sequence of a T. reesei xylanase I is nucleotides 58 to 749 of SEQ ID NO: 23 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 23 encode a signal peptide.
  • the mature polypeptide coding sequence of a T is nucleotides 58 to 2612 of SEQ ID NO: 21 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 21 encode a signal peptide.
  • reesei xylanase II is nucleotides 58 to 778 of SEQ ID NO: 25 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 25 encode a signal peptide.
  • the mature polypeptide coding sequence of a T. reesei xylanase III is nucleotides 49 to 1349 of SEQ ID NO: 27 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 27 encode a signal peptide.
  • the mature polypeptide coding sequence of a T is nucleotides 58 to 778 of SEQ ID NO: 25 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 25 encode a signal peptide.
  • reesei beta-xylosidase is nucleotides 61 to 2391 of SEQ I D NO: 29 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 29 encode a signal peptide.
  • Medium stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
  • Medium-high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more (e.g., several) control sequences.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Polypeptide having cellulolytic enhancing activity means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity.
  • cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1 -50 mg of cellulolytic protein/g of cellulose in PCS).
  • suitable temperature e.g., 50°C, 55°C, or 60°C
  • pH e.g., 5.0 or 5.5
  • a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvaerd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.
  • the GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20- fold.
  • PCS Pretreated corn stover
  • Pretreated Corn Stover means a cellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, or neutral pretreatment.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
  • Subsequence means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having enzyme activity.
  • a subsequence contains at least 85%, e.g., at least 90% or at least 95% of the nucleotides of the mature polypeptide coding sequence of an enzyme.
  • Subtilisin-like serine protease means a protease with a substrate specificity similar to subtilisin that uses a serine residue for catalyzing the hydrolysis of peptide bonds in peptides and proteins.
  • Subtilisin-like proteases are serine proteases characterized by a catalytic triad of the three amino acids aspartate, histidine, and serine. The arrangement of these catalytic residues is shared with the prototypical subtilisin from Bacillus licheniformis (Siezen and Leunissen, 1997, Protein Science 6: 501-523).
  • Subtilisin-like serine protease activity can be determined using a synthetic substrate, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) (Bachem AG, Bubendorf, Switzerland) in 100 mM NaCI-100 mM MOPS pH 7.0 at 50°C for 3 hours and then the absorbance at 405 nm is measured.
  • AAPF N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide
  • Targeted integration means the stable integration of extracellular DNA at a defined genomic locus.
  • Transformant means a cell which has taken up extracellular DNA (foreign, artificial or modified) and expresses the gene(s) contained therein.
  • Transformation means the introduction of extracellular DNA into a cell, i.e., the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
  • Trypsin-like serine protease means a protease with a substrate specificity similar to trypsin that uses a serine residue for catalyzing the hydrolysis of peptide bonds in peptides and proteins.
  • trypsin-like serine protease activity is determined according to the procedure described by Dienes et al., 2007, Enzyme and Microbial Technology 40: 1087- 1094.
  • variant means a polypeptide having enzyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
  • Very high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
  • Very low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
  • xylan-containing material means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)- linked xylose residues.
  • Xylans of terrestrial plants are heteropolymers possessing a beta- (1 -4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose.
  • Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al. , 2005, Adv. Polym. Sci. 186: 1- 67.
  • any material containing xylan may be used.
  • the xylan-containing material is lignocellulose.
  • xylan degrading activity or xylanolytic activity means a biological activity that hydrolyzes xylan-containing material.
  • the two basic approaches for measuring xylanolytic activity include: (1 ) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyi esterases, and alpha-glucuronyl esterases).
  • Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans.
  • the most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.
  • Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 (4-(1 , 1 ,3,3- tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37°C.
  • TRITON® X-100 (4-(1 , 1 ,3,3- tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
  • xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.
  • PBAH p-hydroxybenzoic acid hydrazide
  • xylanase means a 1 ,4-beta-D-xylan-xylohydrolase (E.C.
  • xylanase activity is determined with 0.2% AZCL- arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
  • the present invention relates to enzyme compositions, comprising (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the enzyme compositions further comprise an Aspergillus fumigatus xylanase, an Aspergillus fumigatus beta-xylosidase, or a combination thereof; or homologs thereof.
  • the enzyme compositions of the present invention are more efficient in the deconstruction of cellulosic material than a cellulolytic enzyme composition produced by T. reesei.
  • any Aspergillus fumigatus cellobiohydrolase I, Aspergillus fumigatus cellobiohydrolase II, an Aspergillus fumigatus beta-glucosidase or variant thereof, Penicillium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity, Aspergillus fumigatus xylanase, or Aspergillus fumigatus beta-xylosidase, or homologs thereof, may be used.
  • the Aspergillus fumigatus cellobiohydrolase I or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consist
  • the Aspergillus fumigatus cellobiohydrolase II or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consist
  • the Aspergillus fumigatus beta-glucosidase or a homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6; (iii) a beta-glucosidase encoded by a polynucleotide
  • the Penicillium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity or a homolog thereof is selected from the group consisting of: (i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 8; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii)
  • the Aspergillus fumigatus xylanase or a homolog thereof is selected from the group consisting of: (i) a xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (iii)
  • the Aspergillus fumigatus beta-xylosidase or a homolog thereof is selected from the group consisting of: (i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16; (ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g.
  • the polynucleotide of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding enzymes according to methods well known in the art.
  • probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
  • the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library may be screened for DNA that hybridizes with the probes described above and encodes an enzyme.
  • Genomic or other DNA may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or 21 ; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under very low to very high stringency conditions.
  • Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
  • the nucleic acid probe is SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or the mature polypeptide coding sequence thereof.
  • the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; the mature polypeptide thereof; or a fragment thereof.
  • the techniques used to isolate or clone a polynucleotide include isolation from genomic DNA or cDNA, or a combination thereof.
  • the cloning of the polynucleotides from genomic DNA can be effected, e.g. , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g. , Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA polynucleotide-based amplification
  • the polynucleotides may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
  • a protein engineered variant of an enzyme above may also be used.
  • the variant is an Aspergillus fumigatus beta-glucosidase variant.
  • the A. fumigatus beta-glucosidase variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512 of SEQ ID NO: 6, wherein the variant has beta-glucosidase activity.
  • the variant has sequence identity of at least 80%, e.g.
  • the variant has at least 80%, e.g. , at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 6.
  • the mature polypeptide disclosed in SEQ ID NO: 6 is used to determine the corresponding amino acid residue in another beta- glucosidase.
  • the amino acid sequence of another beta-glucosidase is aligned with the mature polypeptide disclosed in SEQ ID NO: 6, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 6 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • a variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512.
  • a variant comprises a substitution at two positions corresponding to any of positions 100, 283, 456, and 512. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 100, 283, 456, and 512. In another aspect, a variant comprises a substitution at each position corresponding to positions 100, 283, 456, and 512.
  • the variant comprises or consists of a substitution at a position corresponding to position 100.
  • the amino acid at a position corresponding to position 100 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp.
  • the variant comprises or consists of the substitution F100D of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of a substitution at a position corresponding to position 283.
  • the amino acid at a position corresponding to position 283 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gly
  • the variant comprises or consists of the substitution S283G of the mature polypeptide of SEQ I D NO: 6.
  • the variant comprises or consists of a substitution at a position corresponding to position 456.
  • the amino acid at a position corresponding to position 456 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu.
  • the variant comprises or consists of the substitution N456E of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of a substitution at a position corresponding to position 512.
  • the amino acid at a position corresponding to position 512 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Tyr.
  • the variant comprises or consists of the substitution F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of a substitution at positions corresponding to positions 100 and 283, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100 and 456, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100 and 512, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 283 and 456, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 283 and 512, such as those described above. In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 456 and 512, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, and 456, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, and 512, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100, 456, and 512, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 283, 456, and 512, such as those described above.
  • the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, 456, and 512, such as those described above.
  • the variant comprises or consists of one or more (several) substitutions selected from the group consisting of G142S, Q183R, H266Q, and D703G.
  • the variant comprises or consists of the substitutions F100D +
  • the variant comprises or consists of the substitutions F100D + N456E of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions F100D + F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions S283G + N456E of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions S283G + F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions N456E +
  • the variant comprises or consists of the substitutions F100D + S283G + N456E of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions F100D + S283G + F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions F100D + N456E + F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions S283G + N456E + F512Y of the mature polypeptide of SEQ ID NO: 6.
  • the variant comprises or consists of the substitutions F100D +
  • the variants may consist of 720 to 863 amino acids, e.g., 720 to 739, 740 to to 759, 760 to 779, 780 to 799, 800 to 819, 820 to 839, and 840 to 863 amino acids.
  • the variants may further comprise an alteration at one or more (several) other positions.
  • the enzyme composition may further comprise one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
  • One or more (e.g., several) of the enzymes may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins.
  • one or more (e.g., several) enzymes may be native proteins of a cell, which is used as a host cell to express recombinantly the enzyme composition.
  • bacterial endoglucanases examples include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No. 5,536,655, WO 00/70031 , WO 05/093050); T ermobifida fusca endoglucanase III (WO 05/093050); and T ermobifida fusca endoglucanase V (WO 05/093050).
  • an Acidothermus cellulolyticus endoglucanase WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No. 5,536,655, WO 00/70031 , WO 05/093050
  • fungal endoglucanases examples include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al. , 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GENBANKTM accession no. M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:1 1-22), Trichoderma reesei Cel5A endoglucanase II (GENBANKTM accession no.
  • Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555- 563, GENBANKTM accession no. AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, GENBANKTM accession no.
  • the enzyme composition further comprises a Trichoderma endoglucanase I. In another aspect, the enzyme composition further comprises a Trichoderma reesei endoglucanase I. In another aspect, the enzyme composition further comprises a Trichoderma reesei Cel7B endoglucanase I (GENBANKTM accession no. M15665). In another aspect, the Trichoderma reesei endoglucanase I is native to the host cell. In another aspect, the the Trichoderma reesei endoglucanase I is the mature polypeptide of SEQ ID NO: 90.
  • the enzyme composition further comprises a Trichoderma endoglucanase II. In another aspect, the enzyme composition further comprises a Trichoderma reesei endoglucanase II. In another aspect, the enzyme composition further comprises a Trichoderma reesei Cel5A endoglucanase II (GENBANKTM accession no. M19373). In another aspect, the Trichoderma reesei endoglucanase II is native to the host cell. In another aspect, the the Trichoderma reesei endoglucanase I is the mature polypeptide of SEQ ID NO: 92.
  • compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the compositions may be stabilized in accordance with methods known in the art.
  • the enzyme composition may also be a fermentation broth formulation or a cell composition.
  • fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
  • the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g. , filamentous fungal cells) are removed, e.g., by centrifugation.
  • the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
  • the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
  • the fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • the cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis.
  • the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
  • the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • the whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • the present invention also relates to recombinant filamentous fungal host cells, comprising polynucleotides encoding (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • the host cell may be any filamentous fungal cell useful in the recombinant production of an enzyme or protein.
  • “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus,
  • the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zona
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et ai, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • the filamentous fungal cell is any Trichoderma cell useful in the recombinant production of an enzyme or protein.
  • the Trichoderma cell may be a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • the Trichoderma cell is a Trichoderma harzianum cell.
  • the Trichoderma cell is a Trichoderma koningii cell.
  • the Trichoderma cell is a Trichoderma longibrachiatum cell.
  • the Trichoderma cell is a Trichoderma reesei cell.
  • the Trichoderma cell is a Trichoderma viride cell.
  • the Trichoderma reesei cell is Trichoderma reesei RutC30. In another aspect, the Trichoderma reesei cell is Trichoderma reese/ TV10. In another aspect, the Trichoderma reesei cell is a mutant of Trichoderma reesei RutC30. In another aspect, the Trichoderma reesei cell is mutant of Trichoderma reesei TV10. In another aspect, the Trichoderma reesei cell is a morphological mutant of Trichoderma reesei. See, for example, WO 97/26330, which is incorporated herein by reference in its entirety.
  • a Trichoderma cell may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al. , 1988, Bio/Technology 6: 1419-1422.
  • One or more (e.g., several) native cellulase and/or hemicellulase genes may be inactivated in the Trichoderma host cell by disrupting or deleting the genes, or a portion thereof, which results in the mutant cell producing less or none of the cellulase and/or hemicellulase than the parent cell when cultivated under the same conditions.
  • the one or more (e.g., several) cellulase genes encode enzymes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, beta-glucosidase, and swollenin.
  • the one or more (e.g., several) hemicellulase genes encode enzymes selected from the group consisting of xylanase I, xylanase II, xylanase III, and beta-xylosidase.
  • the one or more (e.g., several) hemicellulase genes encode enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, and a mannosidase.
  • enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase
  • the mutant cell may be constructed by reducing or eliminating expression of a polynucleotide encoding a Trichoderma cellulase or hemicellulase using methods well known in the art, for example, insertions, disruptions, replacements, or deletions.
  • the polynucleotide is inactivated.
  • the polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for expression of the coding region.
  • An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the polynucleotide.
  • Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.
  • Modification or inactivation of the polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the polynucleotide has been reduced or eliminated.
  • the mutagenesis which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • MNNG N-methyl-N'-nitro-N- nitrosoguanidine
  • EMS ethyl methane sulphonate
  • sodium bisulphite formic acid
  • nucleotide analogues examples include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleot
  • the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.
  • Modification or inactivation of the polynucleotide may also be accomplished by insertion, substitution, or deletion of one or more (e.g., several) nucleotides in the gene or a regulatory element required for transcription or translation thereof.
  • nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame.
  • modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.
  • the modification may be performed in vivo, i.e., directly on the cell expressing the polynucleotide to be modified, it is preferred that the modification be performed in vitro as exemplified below.
  • An example of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption.
  • a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene.
  • the defective nucleic acid sequence replaces the endogenous polynucleotide.
  • the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed.
  • the polynucleotide is disrupted with a selectable marker such as those described herein.
  • Modification or inactivation of the polynucleotide may also be accomplished by inhibiting expression of an enzyme encoded by the polynucleotide in a cell by administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide encoding the enzyme.
  • dsRNA double-stranded RNA
  • the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
  • the dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA).
  • the dsRNA is small interfering RNA for inhibiting transcription.
  • the dsRNA is micro RNA for inhibiting translation.
  • the double-stranded RNA (dsRNA) molecules comprise a portion of the mature polypeptide coding sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ ID NO: 29 for inhibiting expression of the polypeptide in a cell.
  • the dsRNA can enter a cell and cause the degradation of a single- stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs.
  • ssRNA single- stranded RNA
  • mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).
  • the dsRNAs can be used in gene-silencing to selectively degrade RNA using a dsRNAi of the present invention.
  • the process may be practiced in vitro, ex vivo or in vivo.
  • the dsRNA molecules can be used to generate a loss-of-f unction mutation in a cell, an organ or an animal.
  • Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559; 6,51 1 ,824; and 6,515,109.
  • the Trichoderma cellobiohydrolase I or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 18; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 18; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucle
  • the Trichoderma cellobiohydrolase II or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 20; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 20; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucle
  • the Trichoderma beta-glucosidase or a homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 22; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g.
  • the Trichoderma xylanase or a homolog thereof is selected from the group consisting of: (i) a xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28; (iii) a xylanas
  • the Trichoderma beta-xylosidase or a homolog thereof is selected from the group consisting of: (i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 30; (ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 30; (iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of
  • a Trichoderma cellobiohydrolase I gene is inactivated.
  • a Trichoderma cellobiohydrolase II gene is inactivated.
  • a Trichoderma beta-glucosidase gene is inactivated.
  • a Trichoderma xylanase gene is inactivated.
  • a Trichoderma beta-xylosidase gene is inactivated.
  • Trichoderma cellobiohydrolase I gene and a Trichoderma cellobiohydrolase II gene are inactivated.
  • two or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase are inactivated.
  • three or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
  • four or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
  • five or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta- xylosidase genes are inactivated.
  • six or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta- glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
  • the cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
  • one or more (e.g., several) protease genes are inactivated.
  • the one or more (e.g., several) protease genes are subtilisin-like serine protease, aspartic protease, and trypsin-like serine protease genes as described in WO 201 1/075677, which is incorporated herein by reference in its entirety.
  • Nucleic acid constructs comprising a polynucleotide encoding an enzyme or protein can be constructed by operably linking one or more (e.g., several) control sequences to the polynucleotide to direct the expression of the coding sequence in a filamentous fungal host cell under conditions compatible with the control sequences.
  • Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector.
  • the techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a filamentous fungal host cell for expression of a polynucleotide encoding an enzyme or protein.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • promoters for directing transcription of the nucleic acid constructs in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase ⁇ glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00
  • the control sequence may also be a transcription terminator, which is recognized by a filamentous fungal host cell to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin- like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase II I, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma re
  • the control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by a filamentous fungal host cell.
  • the leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
  • Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by a filamentous fungal host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichoderma reesei endoglucanase V.
  • the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into a cell's secretory pathway.
  • the 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
  • the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
  • a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide.
  • any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, and Trichoderma reesei endoglucanase V.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Myceliophthora thermophila laccase (WO 95/33836) and Rhizomucor miehei aspartic proteinase.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • regulatory sequences that regulate expression of the polypeptide relative to the growth of a filamentous fungal host cell.
  • regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory sequences include the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter.
  • Other examples of regulatory sequences are those that allow for gene amplification.
  • these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.
  • the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.
  • Recombinant expression vectors can be constructed comprising a polynucleotide encoding an enzyme or protein, a promoter, a terminator, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • adeA phosphoribosylaminoimidazole-succinocarboxamide synthase
  • adeB phosphorib
  • Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
  • Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
  • the selectable marker may be a dual selectable marker system as described in WO
  • the selectable marker is a hph-tk dual selectable marker system.
  • the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in a filamentous fungal host cell.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term "origin of replication" or "plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • AMA1 and ANSI examples of origins of replication useful in a filamentous fungal host cell are AMA1 and ANSI (Gems ef a/., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
  • More than one copy of a polynucleotide may be inserted into a filamentous fungal host cell to increase production of a polypeptide.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to methods of producing an enzyme composition, comprising: (a) cultivating a filamentous fungal host cell of the present invention under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
  • the filamentous fungal host cells are cultivated in a nutrient medium suitable for production of the enzyme composition using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the enzymes to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
  • Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
  • the enzymes may be detected using methods known in the art that are specific for the enzyme. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine activity.
  • the enzymes may be recovered using methods known in the art.
  • the enzyme may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the whole fermentation broth is recovered.
  • the enzymes may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to
  • the present invention is also directed to the following processes for using an enzyme composition of the present invention.
  • the present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition of the present invention.
  • the processes further comprise recovering the degraded or converted cellulosic material.
  • Soluble products of degradation or conversion of the cellulosic material can be separated from insoluble cellulosic material using a method known in the art such as, for example, centrifugation, filtration, or gravity settling.
  • the present invention also relates to processes of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • the present invention also relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition of the present invention.
  • the fermenting of the cellulosic material produces a fermentation product.
  • the processes further comprise recovering the fermentation product from the fermentation.
  • the processes of the present invention can be used to saccharify the cellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids, alcohols, ketones, gases, and the like).
  • useful fermentation products e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids, alcohols, ketones, gases, and the like).
  • platform chemicals e.g., acids, alcohols, ketones, gases, and the like.
  • the processing of the cellulosic material according to the present invention can be accomplished using methods conventional in the art. Moreover, the processes of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
  • Hydrolysis (saccharification) and fermentation, separate or simultaneous include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP).
  • SHF uses separate process steps to first enzymatically hydrolyze the cellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol.
  • SSF the enzymatic hydrolysis of the cellulosic material and the fermentation of sugars to ethanol are combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212).
  • SSCF involves the co-fermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827).
  • HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor.
  • the steps in an HHF process can be carried out at different temperatures, i.e. , high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate.
  • DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I.
  • a conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol.
  • an attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive stirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153).
  • Additional reactor types include fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.
  • any pretreatment process known in the art can be used to disrupt plant cell wall components of the cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?, Adv. Biochem. Engin. /Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin. /Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol.
  • the cellulosic material can also be subjected to particle size reduction, sieving, pre- soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.
  • Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment.
  • Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical C0 2 , supercritical H 2 0, ozone, ionic liquid, and gamma irradiation pretreatments.
  • the cellulosic material can be pretreated before hydrolysis and/or fermentation. Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).
  • the cellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g. , hemicellulose, accessible to enzymes.
  • the cellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time.
  • Steam pretreatment is preferably performed at 140-250°C, e.g. , 160-200°C or 170-190°C, where the optimal temperature range depends on addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1 -60 minutes, e.g.
  • Steam pretreatment allows for relatively high solids loadings, so that the cellulosic material is generally only moist during the pretreatment.
  • the steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 20020164730).
  • hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.
  • Chemical Pretreatment refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose.
  • suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments.
  • a catalyst such as H 2 S0 4 or S0 2 (typically 0.3 to 5% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al. , 2006, Appl. Biochem. Biotechnol. 129- 132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 1 13-1 16: 509-523; Sassner et al. , 2006, Enzyme Microb. Technol. 39: 756-762).
  • the cellulosic material is mixed with dilute acid, typically H 2 S0 4 , and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure.
  • dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91 : 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-1 15).
  • alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).
  • Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150°C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673- 686).
  • WO 2006/1 10891 , WO 2006/1 10899, WO 2006/1 10900, and WO 2006/1 10901 disclose pretreatment methods using ammonia.
  • Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151 ; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81 : 1669-1677).
  • the pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.
  • Ammonia fiber explosion involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-150°C and high pressure such as 17- 20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231 ; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121 : 1 133-1 141 ; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018).
  • cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.
  • Organosolv pretreatment delignifies the cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200°C for 30-60 minutes (Pan et al. , 2005, Biotechnol. Bioeng. 90: 473-481 ; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861 ; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121 : 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.
  • the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment.
  • the acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.
  • Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5.
  • the acid concentration is in the range from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt % acid.
  • the acid is contacted with the cellulosic material and held at a temperature in the range of preferably 140-200°C, e.g., 165-190°C, for periods ranging from 1 to 60 minutes.
  • pretreatment takes place in an aqueous slurry.
  • the cellulosic material is present during pretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt % or 30-60 wt %, such as around 40 wt %.
  • the pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
  • mechanical pretreatment or Physical pretreatment refers to any pretreatment that promotes size reduction of particles.
  • pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
  • the cellulosic material can be pretreated both physically (mechanically) and chemically.
  • Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof.
  • high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi.
  • high temperature means temperatures in the range of about 100 to about 300°C, e.g., about 140 to about 200°C.
  • mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
  • the physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
  • the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.
  • Biopretreatment refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulosic material.
  • Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass, Adv. Appl. Microbiol.
  • Saccharification In the hydrolysis step, also known as saccharification, the cellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides.
  • the hydrolysis is performed enzymatically by an enzyme composition of the present invention.
  • Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art.
  • hydrolysis is performed under conditions suitable for the activity of the enzyme(s), i.e., optimal for the enzyme(s).
  • the hydrolysis can be carried out as a fed batch or continuous process where the cellulosic material is fed gradually to, for example, an enzyme containing hydrolysis solution.
  • the saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art.
  • the saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 120 hours, e.g., about 16 to about 72 hours or about 24 to about 48 hours.
  • the temperature is in the range of preferably about 25°C to about 70°C, e.g., about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to about 55°C.
  • the pH is in the range of preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5.
  • the dry solids content is in the range of preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt % or about 20 to about 30 wt %.
  • the enzyme composition of the present invention can be added prior to or during fermentation, e.g., during saccharification or during or after propagation of the fermenting microorganism(s).
  • the enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a Trichoderma host cell as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • the optimum amount of Aspergillus fumigatus cellulases or hemicellulases depends on several factors including, but not limited to, the mixture of component cellulolytic and/or hemicellulolytic enzymes, the cellulosic material, the concentration of cellulosic material, the pretreatment(s) of the cellulosic material, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).
  • fermenting organism e.g., yeast for Simultaneous Saccharification and Fermentation.
  • an effective amount of cellulolytic or hemicellulolytic enzyme to the cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1 .25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1 .25 mg, about 0.15 to about 1 .25 mg, or about 0.25 to about 1.0 mg per g of the cellulosic material.
  • the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, e.g., manganese sulfate.
  • a GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated cellulosic material such as pretreated corn stover (PCS).
  • PCS pretreated corn stover
  • the dioxy compound may include any suitable compound containing two or more oxygen atoms.
  • the dioxy compounds contain a substituted aryl moiety as described herein.
  • the dioxy compounds may comprise one or more (e.g., several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives.
  • Non-limiting examples of the dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1 ,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6- dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1 ,2-benzenediol; 4- nitro-1 ,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2- butyne-1 ,4-diol; (croconic acid; 1 ,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1 ,2- propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-buten
  • the bicyclic compound may include any suitable substituted fused ring system as described herein.
  • the compounds may comprise one or more (e.g., several) additional rings, and are not limited to a specific number of rings unless otherwise stated.
  • the bicyclic compound is a flavonoid.
  • the bicyclic compound is an optionally substituted isoflavonoid.
  • the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof.
  • Non-limiting examples of the bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.
  • the heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein.
  • the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety.
  • the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5-membered heterocycloalkyl or an optionally substituted 5- membered heteroaryl moiety.
  • the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl,
  • the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl.
  • the heterocyclic compounds include (1 ,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3-furanone; 5- hydroxy-2(5H)-furanone; [1 ,2-dihydroxyethyl]furan-2,3,4(5H)-trione; ohydroxy-v- butyrolactone; ribonic ⁇ -lactone; aldohexuronicaldohexuronic acid ⁇ -lactone; gluconic acid ⁇ - lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)- furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one
  • the nitrogen-containing compound may be any suitable compound with one or more (e.g., several) nitrogen atoms.
  • the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety.
  • Non-limiting examples of the nitrogen- containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2- aminophenol; 1 ,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8- tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.
  • the quinone compound may be any suitable compound comprising a quinone moiety as described herein.
  • the quinone compounds include 1 ,4- benzoquinone; 1 ,4-naphthoquinone; 2-hydroxy-1 ,4-naphthoquinone; 2,3-dimethoxy-5- methyl-1 ,4-benzoquinone or coenzyme Q 0 ; 2,3,5,6-tetramethyl-1 ,4-benzoquinone or duroquinone; 1 ,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1 ,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.
  • the sulfur-containing compound may be any suitable compound comprising one or more (e.g., several) sulfur atoms.
  • the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester.
  • Non-limiting examples of the sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1 -thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene- 1 ,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.
  • an effective amount of such a compound described above to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10 "6 to about 10, e.g., about 10 "6 to about 7.5, about 10 "6 to about 5, about 10 "6 to about 2.5, about 10 "6 to about 1 , about 10 "5 to about 1 , about 10 "5 to about 10 “1 , about 10 “4 to about 10 "1 , about 10 "3 to about 10 “1 , or about 10 "3 to about 10 "2 .
  • an effective amount of such a compound described above is about 0.1 ⁇ to about 1 M, e.g., about 0.5 ⁇ to about 0.75 M, about 0.75 ⁇ to about 0.5 M, about 1 ⁇ to about 0.25 M, about 1 ⁇ to about 0.1 M, about 5 ⁇ to about 50 mM, about 10 ⁇ to about 25 mM, about 50 ⁇ to about 25 mM, about 10 ⁇ to about 10 mM, about 5 ⁇ to about 5 mM, or about 0.1 mM to about 1 mM.
  • liquid means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, eic , under conditions as described herein, and the soluble contents thereof.
  • a liquor for cellulolytic enhancement of a GH61 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids.
  • a catalyst e.g., acid
  • organic solvent optionally in combination with physical disruption of the material
  • Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate by a cellulase preparation.
  • the liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.
  • an effective amount of the liquor to cellulose is about 10 "6 to about 10 g per g of cellulose, e.g., about 10 "6 to about 7.5 g, about 10 "6 to about 5, about 10 "6 to about 2.5 g, about 10 "6 to about 1 g, about 10 "5 to about 1 g, about 10 "5 to about 10 "1 g, about 10 “4 to about 10 "1 g, about 10 "3 to about 10 "1 g, or about 10 "3 to about 10 "2 g per g of cellulose.
  • the fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product.
  • Fermentation or “fermentation process” refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry.
  • the fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
  • sugars released from the cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast.
  • Hydrolysis (saccharification) and fermentation can be separate or simultaneous, as described herein.
  • Any suitable hydrolyzed cellulosic material can be used in the fermentation step in practicing the present invention.
  • the material is generally selected based on the desired fermentation product, i.e. , the substance to be obtained from the fermentation, and the process employed, as is well known in the art.
  • fermentation medium is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
  • SSF simultaneous saccharification and fermentation process
  • “Fermenting microorganism” refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product.
  • the fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art.
  • Suitable fermenting microorganisms are able to ferment, i.e. , convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al, 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
  • yeast examples include bacterial and fungal organisms, such as yeast.
  • Preferred yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
  • Preferred xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, preferably P. stipitis, such as P. stipitis CBS 5773.
  • Preferred pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus.
  • Organisms not capable of fermenting pentose sugars, such as xylose and arabinose may be genetically modified to do so by methods known in the art.
  • fermenting organisms include strains of Bacillus, such as Bacillus coagulans;
  • Candida such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;
  • Klebsiella such as K. oxytoca
  • Kluyveromyces such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis
  • Schizosaccharomyces such as S. pombe
  • Thermoanaerobacter such as Thermoanaerobacter saccharolyticum
  • Zymomonas such as Zymomonas mobilis.
  • the yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis.
  • the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia.
  • the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces spp. In another more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.
  • the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacilus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.
  • yeast suitable for ethanol production include, e.g., BIOFERMTM AFT and XR (NABC - North American Bioproducts Corporation, GA, USA), ETHANOL REDTM yeast (Fermentis/Lesaffre, USA), FALITM (Fleischmann's Yeast, USA), FERMIOLTM (DSM Specialties), GERT STRANDTM (Gert Strand AB, Sweden), and SUPERSTARTTM and THERMOSACCTM fresh yeast (Ethanol Technology, Wl, USA).
  • BIOFERMTM AFT and XR NABC - North American Bioproducts Corporation, GA, USA
  • ETHANOL REDTM yeast Fermentis/Lesaffre, USA
  • FALITM Ferischmann's Yeast, USA
  • FERMIOLTM DSM Specialties
  • GERT STRANDTM Gert Strand AB, Sweden
  • SUPERSTARTTM and THERMOSACCTM fresh yeast Ethanol Technology, Wl, USA.
  • the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
  • the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.
  • the fermenting microorganism is typically added to the degraded cellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g., about 24 to about 60 hours.
  • the temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
  • the yeast and/or another microorganism are applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours.
  • the temperature is preferably between about 20°C to about 60°C, e.g. , about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C
  • the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7.
  • some fermenting organisms, e.g., bacteria have higher fermentation temperature optima.
  • Yeast or another microorganism is preferably applied in amounts of approximately 10 5 to 10 12 , preferably from approximately 10 7 to 10 10 , especially approximately 2 x 10 8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
  • a fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield.
  • a "fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast.
  • Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E.
  • minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
  • a fermentation product can be any substance derived from the fermentation.
  • the fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1 ,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g.
  • an alcohol e.g., arabinitol, n-butanol, isobutanol
  • pentene, hexene, heptene, and octene an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H 2 ), carbon dioxide (C0 2 ), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D- gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and x
  • the fermentation product is an alcohol.
  • alcohol encompasses a substance that contains one or more (e.g., several) hydroxyl moieties.
  • the alcohol is n-butanol.
  • the alcohol is isobutanol.
  • the alcohol is ethanol.
  • the alcohol is methanol.
  • the alcohol is arabinitol.
  • the alcohol is butanediol.
  • the alcohol is ethylene glycol.
  • the alcohol is glycerin.
  • the alcohol is glycerol.
  • the alcohol is 1 ,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer- Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol.
  • the fermentation product is an alkane.
  • the alkane can be an unbranched or a branched alkane.
  • the alkane is pentane.
  • the alkane is hexane.
  • the alkane is heptane.
  • the alkane is octane.
  • the alkane is nonane.
  • the alkane is decane.
  • the alkane is undecane.
  • the alkane is dodecane.
  • the fermentation product is a cycloalkane.
  • the cycloalkane is cyclopentane.
  • the cycloalkane is cyclohexane.
  • the cycloalkane is cycloheptane.
  • the cycloalkane is cyclooctane.
  • the fermentation product is an alkene.
  • the alkene can be an unbranched or a branched alkene.
  • the alkene is pentene.
  • the alkene is hexene.
  • the alkene is heptene.
  • the alkene is octene.
  • the fermentation product is an amino acid.
  • the organic acid is aspartic acid.
  • the amino acid is glutamic acid.
  • the amino acid is glycine.
  • the amino acid is lysine.
  • the amino acid is serine.
  • the amino acid is threonine. See, for example, Richard, A., and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.
  • the fermentation product is a gas.
  • the gas is methane. In another more preferred aspect, the gas is H 2 . In another more preferred aspect, the gas is C0 2 . In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V.N. in Biomass and Bioenergy, Vol. 13 (1 -2), pp. 83-1 14, 1997, Anaerobic digestion of biomass for methane production: A review.
  • the fermentation product is isoprene.
  • the fermentation product is a ketone.
  • ketone encompasses a substance that contains one or more (e.g., several) ketone moieties.
  • the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
  • the fermentation product is an organic acid.
  • the organic acid is acetic acid.
  • the organic acid is acetonic acid.
  • the organic acid is adipic acid.
  • the organic acid is ascorbic acid.
  • the organic acid is citric acid.
  • the organic acid is 2,5-diketo-D-gluconic acid.
  • the organic acid is formic acid.
  • the organic acid is fumaric acid.
  • the organic acid is glucaric acid.
  • the organic acid is gluconic acid.
  • the organic acid is glucuronic acid.
  • the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448.
  • the fermentation product is polyketide
  • the fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction.
  • alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • the present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
  • Trichoderma reesei strain 981-0-8 (D4) is a mutagenized strain of Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181 : 289- 301 ).
  • Trichoderma reesei strain AgJgl 15-104-7B1 (PCT/US2010/061 105; WO 201 1/075677) is a T. reesei ku70- derivative of strain 981-0-8 (D4).
  • 2XYT plus ampicillin plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of sodium chloride, 15 g of Bacto agar, and deionized water to 1 liter. One ml of a 100 mg/ml solution of ampicillin was added after the autoclaved medium was cooled to 55 ° C.
  • SOC medium was composed of 20 g of Bacto-tryptone, 5 g of Bacto yeast extract, 0.5 g of NaCI, 2.5 ml of 1 M KCI, and deionized water to 1 liter. The pH was adjusted to 7.0 with 10 N NaOH before autoclaving. Then 20 ml of sterile 1 M glucose was added immediately before use.
  • COVE salt solution was composed of 26 g of KCI, 26 g of MgS0 4 -7H 2 0, 76 g of
  • COVE trace metals solution was composed of 0.04 g of NaB 4 O 7 -10H 2 O, 0.4 g of
  • COVE plates were composed of 342.3 g of sucrose, 20 ml of COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCI, 25 g of Noble agar (Difco), and deionized water to
  • COVE2 plates were composed of 30 g of sucrose, 20 ml of COVE salt solution, 10 ml of 1 M acetamide, 25 g of Noble agar (Difco), and deionized water to 1 liter.
  • Trichoderma trace metals solution was composed of 216 g of FeCI 3 -6H 2 0, 58 g of
  • CIM medium was composed of 20 g of cellulose, 10 g of corn steep solids, 1 .45 g of (NH 4 ) 2 S0 4 , 2.08 g of KH 2 P0 4 , 0.28 g of CaCI 2 , 0.42 g of MgS0 4 -7H 2 0, 0.42 ml of Trichoderma trace metals solution, 1-2 drops of antifoam, and deionized water to 1 liter; pH adjusted to 6.0.
  • YP medium was composed of 10 g of yeast extract, 20 g of Bacto peptone, and deionized water to 1 liter.
  • PEG buffer was composed of 500 g of polyethylene glycol 4000 (PEG 4000), 10 mM CaCI 2 , 10 mM Tris-HCI pH 7.5, and deionized water to 1 liter; filter sterilized.
  • PDA plates were composed of 39 g of Potato Dextrose Agar (Difco) and deionized water to 1 liter.
  • PDA overlay medium was composed of 39 g of Potato Dextrose Agar (Difco), 2.44 g uridine, and deionized water to 1 liter. The previously autoclaved medium was melted in a microwave and then tempered to 55°C before use.
  • STC was composed of 1 M sorbitol, 10 mM mM CaCI 2 , and 10 mM Tris-HCI, pH 7.5; filter sterilized.
  • TE buffer was composed of 1 M Tris pH 8.0 and 0.5 M EDTA pH 8.0.
  • 20X SSC was composed of 175.3 g of NaCI, 88.2 g of sodium citrate, and deionized water to 1 liter.
  • TrMM-G medium was composed of 20 ml of COVE salt solution, 6 g of (NH 4 ) 2 S0 , 0.6 g of CaCI 2 , 25 g of Nobel agar (Difco), 20 g of glucose, and deionized water to 1 liter.
  • NZY+ medium was composed of 5 g of NaCI, 3 g of MgS0 4 -7H 2 0, 5 g of yeast extract, 10 g of NZ amine, 1.2 g of MgCI 2 , 4 g of glucose, and deionized water to 1 liter.
  • Example 1 Construction of a Trichoderma reesei cbhl-Aspergillus fumigatus cbhl replacement construct pJfySI 39
  • the Aspergillus fumigatus cellobiohydrolase I (cbhl) coding sequence (SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid sequence]) was amplified from pEJG93 (WO 201 1/057140) using the gene-specific forward and reverse primers shown below.
  • the region in italics represents vector homology to the site of insertion for an I N- FUSION® reaction and the underlined portion is an introduced Pac I site.
  • the amplification reaction was composed of 20 ng of pEJG93, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X HERCULASE® Reaction Buffer (Stratagene, La Jolla, CA, USA), and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA, USA) in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S (Eppendorf Scientific, Inc., Westbury, NY, USA) programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 7 minutes.
  • EPPENDORF® MASTERCYCLER® 5333 epgradient S Eppendorf Scientific, Inc., Westbury, NY, USA
  • PCR products were separated by 1 % agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) buffer where a 1.6 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol.
  • TAE disodium EDTA
  • the 1.6 kb PCR product was inserted into Nco ⁇ IPac l-digested pSMai155 (WO 05/074647) using an IN-FUSION® Advantage PCR Cloning Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol.
  • the IN-FUSION® reaction was composed of 1X I N-FUSION® Reaction Buffer (Clontech, Palo Alto, CA, USA), 125 ng of Nco ⁇ IPac I- digested pSMai155, 100 ng of the 1.6 kb PCR product, and 1 ⁇ of IN-FUSION® Enzyme (Clontech, Palo Alto, CA, USA) in a 10 ⁇ reaction volume.
  • the reaction was incubated for 15 minutes at 37°C followed by 15 minutes at 50°C. After the incubation period 40 ⁇ of TE buffer were added to the reaction. A 2 ⁇ aliquot was used to transform ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • Plasmid pJfyS139-A was used for insertion of the Herpes simplex virus thymidine kinase (tk) gene.
  • the Herpes simplex virus tk coding sequence (SEQ ID NO: 33 [DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) was liberated from pJfySI 579-8-6 (WO 2010/039840) by digesting the plasmid with Bgl II and Bam HI. The digestion was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 2.3 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the tk gene cassette was inserted into Bam Hl-digested, calf intestine phosphatase-treated pJfyS139-A using a QUICK LIGATIONTM Kit (New England Biolabs, Inc., Ipswich, MA USA) according to the manufacturer's protocol.
  • the ligation reaction was composed of 50 ng of the Bam Hl- digested, calf intestine phosphatase-treated pJfyS139-A, 50 ng of the 2.3 kb tk gene insert, 1 X QUICK LIGATIONTM Buffer (New England Biolabs, Inc., Ipswich, MA USA), and 5 units of QUICK LIGASETM (New England Biolabs, Inc., Ipswich, MA USA) in a final volume of 20 ⁇ .
  • the reaction was incubated at room temperature for 5 minutes and 2 ⁇ of the reaction were used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol.
  • the cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Xma I to determine the presence and orientation of the insert and a clone containing the insert was identified and designated pJfyS139-B. Plasmid pJfyS139-B was used for insertion of a T. reesei 3' cbhl gene flanking sequence.
  • the 3' cbhl gene flanking sequence was amplified from T. reesei RutC30 genomic DNA using the forward and reverse primers below.
  • the underlined portion represents an introduced Not I site for cloning.
  • Trichoderma reesei RutC30 was grown in 50 ml of YP medium supplemented with 2% glucose (w/v) in a 250 ml baffled shake flask at 28°C for 2 days with agitation at 200 rpm.
  • Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, La Jolla, CA, USA), washed twice in deionized water, and frozen under liquid nitrogen. Frozen mycelia were ground by mortar and pestle to a fine powder. Total DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia, CA, USA) with the lytic incubation extended to 2 hours.
  • the amplification reaction was composed of 150 ng of T. reesei RutC30 genomic DNA, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes.
  • the PCR reaction was subjected to a MINELUTE® Nucleotide Removal Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol.
  • the resulting PCR mixture was digested with Not I and the digested PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer.
  • a 1.3 kb fragment containing the 3' cbhl gene flanking sequence was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the 1 .3 kb fragment was inserted into Not l-linearized, calf intestine phosphatase-treated pJfyS139-B using a QUICK LIGATIONTM Kit.
  • the QUICK LIGATIONTM reaction was composed of 100 ng of the Not l-linearized, calf intestine phosphatase-treated pJfyS139-B, 20 ng of the 1.3 kb fragment, 1 X QUICK LIGATIONTM Buffer, and 5 units of QUICK LIGASETM in a final volume of 20 ⁇ .
  • the reaction was incubated at room temperature for 5 minutes and 2 ⁇ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Xma I to determine the presence and orientation of the insert and positive clones were sequenced.
  • a clone containing the 3' cbhl gene flanking sequence with no PCR errors was designated pJfyS139 ( Figure 1 ). Plasmid pJfyS139 was used as the vector to replace the T. reesei cbhl gene.
  • Trichoderma reesei strain AgJgl 15-104- 7B1 (PCT/US2010/061 105, WO 201 1/075677) was cultivated in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine at 27°C for 17 hours with gentle agitation at 90 rpm.
  • Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System (Millipore, Bedford, MA, USA) and washed twice with deionized water and twice with 1.2 M sorbitol.
  • Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX® 200 G (Novozymes A/S, Bagsvaerd, Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St. Louis, MO, USA) per ml for 15-25 minutes at 34°C with gentle shaking at 90 rpm.
  • Protoplasts were collected by centrifuging for 7 minutes at 400 x g and washed twice with cold 1 .2 M sorbitol. The protoplasts were counted using a haemocytometer and re-suspended to a final concentration of 1x10 8 protoplasts per ml in STC. Excess protoplasts were stored in a Cryo 1 °C Freezing Container (Nalgene, Rochester, NY, USA) at -80°C.
  • the digestion reaction was purified by 1 % agarose gel electrophoresis using TAE buffer.
  • a DNA band was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA).
  • the resulting purified DNA was added to 100 ⁇ of the protoplast solution and mixed gently.
  • PEG buffer 250 ⁇
  • STC (3 ml) was then added, mixed, and spread onto PDA plates supplemented with 1 M sucrose.
  • Example 3 Replacement of the native Trichoderma reesei cbhl gene with the Aspergillus fumigatus cbhl coding sequence
  • Trichoderma reesei native cbhl gene SEQ ID NO: 17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid sequence]
  • Trichoderma reesei ku70- strain AgJgl 15-104-7B1 PCT/US2010/061 105, WO 201 1/075677
  • was transformed with 4 x 2 ⁇ g of Pme l-linearized pJfyS139 Example 1
  • Seven transformants were obtained and each one was picked and transferred to a PDA plate and incubated for 7 days at 28°C. Genomic DNA was isolated from the transformants according to the procedure described in Example 1 and each transformant submiited to Southern analysis.
  • the DNA was UV crosslinked to the membrane using a STRATALINKERTM UV Crosslinker (Stratagene, La Jolla, CA, USA) and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb (Roche Diagnostics Corporation, Indianapolis, IN, USA).
  • a probe hybridizing to the 3' cbhl gene flanking sequence was generated using a PCR Dig Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions with the forward and reverse primers shown below.
  • the PCR reaction was composed of 1X HERCULASE® Reaction Buffer, 400 nM of each primer, 200 ⁇ DIG-labeled dUTP-containing dNTPs, 20 ng of pJfyS139, and 1.5 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase.
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds; and 1 cycle at 72°C for 7 minutes.
  • the probe was purified by 1 % agarose gel electrophoresis using TAE buffer where a 0.5 kb band corresponding to the probe was excised from the gel and extracted using a MI NELUTE® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42°C for 15-17 hours.
  • the membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C.
  • the probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that 3 of the 7 transformants contained the replacement cassette at the cbhl locus and one transformant, T. reesei JfyS 139-8, was chosen for curing the hpt and tk markers.
  • a fresh plate of spores was generated by transferring spores of a 7 day old PDA plate grown at 28°C to a PDA plate and incubating for 7 days at 28°C. Spores were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemocytometer and 10 5 spores were spread onto 150 mm plates containing TrMM-G medium supplemented with 1 ⁇ 5-fluoro-2'-deoxyuridine (FdU).
  • FdU 5-fluoro-2'-deoxyuridine
  • T reesei cbhll promoter was first amplified from T reesei RutC30 genomic DNA using the gene-specific forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion in an INFUSION® reaction. T reesei RutC30 genomic DNA was prepared according to the procedure described in Example 1.
  • Reverse primer 5'-cgcgiagaicigcggccaiGGTGCAATACACAGAGGGTGATCTT-3' (SEQ ID NO: 40)
  • the amplification reaction was composed of 20 ng of T. reesei RutC30 genomic DNA, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes.
  • the PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 1 .6 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 125 ng of the Nco ⁇ ISal l-digested pSMai155, 100 ng of the 1.6 kb PCR product, and 1 ⁇ of IN-FUSION® Enzyme in a 10 ⁇ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction. A 2 ⁇ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol.
  • the cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Pci I and positive clones sequenced to ensure the absence of PCR errors.
  • One clone containing the insert with no PCR errors was identified and designated pJfyS142-A. Plasmid pJfyS142-A was used to insert the T. reesei cbhll terminator.
  • the cbhll terminator was amplified from T. reesei RutC30 genomic DNA using the gene-specific forward and reverse primers shown below.
  • the region in italics represents vector homology to the site of insertion in an IN-FUSION® reaction.
  • the amplification reaction was composed of 150 ng of T. reesei RutC30 genomic DNA, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 50 seconds; and 1 cycle at 72°C for 7 minutes.
  • PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 0.3 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the 0.3 kb PCR product was inserted into Pac ⁇ IBam Hl-digested pJfyS142-A using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol.
  • the IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 150 ng of the Pacl/Bam Hl-digested pJfyS142-A, 50 ng of the 0.3 kb PCR product, and 1 ⁇ of INFUSION® Enzyme in a 10 ⁇ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction.
  • a 2 ⁇ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol.
  • the cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the transformants were screened by sequence analysis to identify positive clones and to ensure the absence of PCR errors.
  • One clone containing the insert with no PCR errors was identified and designated pJfyS142-B. Plasmid pJfyS142-B was used for insertion of the Herpes simplex tk gene.
  • the Herpes simplex tk gene was liberated from pJfySI 579-8-6 (WO 2010/039840) by digesting the plasmid with Bgl II and Bam HI. The digestion was submitted to 1 % agarose gel electrophoresis using TAE buffer where a 2.3 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The tk cassette was inserted into Bam Hl- digested, calf Intestine phoshatase-dephosphorylated pJfyS142-B using a QUICK LIGATIONTM Kit according to the manufacturer's protocol.
  • the ligation reaction was composed of 50 ng of the Bam Hl-digested, calf Intestine phoshatase-dephosphorylated pJfyS142-B, 50 ng of the 2.3 kb ⁇ / gene insert, 1 X QUICK LIGATIONTM Buffer, and 5 units of QUICK LIGASETM in a 20 ⁇ ligation volume.
  • the reaction was incubated at room temperature for 5 minutes and 2 ⁇ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Xma I and Bam HI to determine the presence and orientation of the insert and a clone containing the insert was identified and designated pJfyS142-C. Plasmid pJfyS142-C was used for insertion of the T. reesei 3' cbhll gene flanking sequence.
  • the 3' cbhll gene flanking sequence was amplified from T. reesei RutC30 genomic
  • the amplification reaction was composed of 150 ng of T. reesei RutC30 genomic
  • DNA 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute 50 seconds; and 1 cycle at 72°C for 7 minutes.
  • the PCR reaction was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 1 .5 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the 3' cbhll gene flanking sequence was inserted into Not l-linearized pJfyS142-C using an INFUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol.
  • the IN- FUSION® reaction was composed of 1 X IN-FUSION® Reaction Buffer, 150 ng of pJfyS142- C, 80 ng of the 1.5 kb PCR product, and 1 ⁇ of IN-FUSION® Enzyme in a 10 ⁇ reaction volume.
  • the reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction. A 2 ⁇ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Bgl II and positive clones were sequenced to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pJfyS142 ( Figure 2). Plasmid pJfyS142 was used to insert the A. fumigatus cbhll coding sequence.
  • the Aspergillus fumigatus cbhll coding sequence (SEQ ID NO: 3 [DNA sequence] and SEQ ID NO: 4 [deduced amino acid sequence]) was amplified from pAILo33 (WO 201 1/057140) using the forward and reverse primers shown below.
  • the region in italics represents vector homology to the site of insertion for an IN-FUSION® reaction.
  • the amplification reaction was composed of 20 ng of pAILo33, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1 mM HERCULASE® Reaction Buffer, and 1.875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes; and 1 cycle at 72°C for 7 minutes.
  • the PCR reaction was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 1.7 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the 1 .7 kb PCR product was inserted into Nco ⁇ IPac l-digested pJfyS142 (Example 4) using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol.
  • the IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 120 ng of the Nco ⁇ IPac l-digested pJfyS142, 70 ng of the 1.7 kb PCR product, and 1 ⁇ of IN-FUSION® Enzyme in a 10 ⁇ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction. A 2 ⁇ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were sequenced to ensure the absence of PCR errors and determine the presence of the insert.
  • One clone with error-free sequence was identified and designated pJfyS144 ( Figure 3). Plasmid pJfyS144 was used to replace the native cbhll gene with the cbhll coding sequence from A. fumigatus.
  • Trichoderma reesei JfyS139-8A (Example 3) was transformed according to the procedure described in Example 2 with 2 ⁇ g of Pme l-linearized and gel purified pJfyS144 (Example 5). Seven transformants were obtained and each one was picked and transferred to a PDA plate and incubated for 7 days at 28°C.
  • a fungal spore PCR method described below was used to screen for transformants bearing gene replacement using the forward primer shown below annealing to a region upstream of the 5' cbhll gene flanking sequence beyond the region of integration, and the reverse primer shown below annealing in the A. fumigatus cbhll coding sequence.
  • a 1.8 kb PCR product would be generated only upon the occurrence of a precise gene replacement at the cbhll locus. If the cassette had integrated elsewhere in the genome, no amplification would result.
  • a small amount of spores from each transformant was suspended in 25 ⁇ of TE buffer and heated on high in a microwave oven for 1 minute.
  • Each microwaved spore suspension was used as a template in the PCR reaction.
  • the reaction was composed of 1 ⁇ of the microwaved spore suspension, 1 ⁇ of a 10 mM dNTPs, 12.5 ⁇ of 2X ADVANTAGE® GC-Melt LA Buffer (Clontech, Mountain View, CA, USA), 25 pmol of forward primer, 25 pmol of reverse primer, 1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix (Clontech, Mountain View, CA, USA), and 9.25 ⁇ of water.
  • the reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 35 cycles each at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute 40 seconds; 1 cycle at 72°C for 7 minutes; and a 4°C hold.
  • the PCR reactions were subjected to 1 % agarose gel electrophoresis using TAE buffer.
  • the spore PCR indicated that four of the seven transformants contained the replacement cassette at the targeted locus and three of them were submitted to Southern analysis to confirm the replacement cassette was in a single copy.
  • Genomic DNA was isolated from the three transformants according to the procedure described in Example 1 and each transformant submitted to Southern analysis.
  • 2 ⁇ g of genomic DNA was digested with 50 units of Dra I in a 50 ⁇ reaction volume and subjected to 1 % agarose electrophoresis in TAE buffer.
  • the DNA in the gel was depurinated with one 10 minute wash in 0.25 N HCI, denatured with two 15 minute washes in 0.5 N NaOH-1 .5 M NaCI, neutralized with one 30 minute wash in 1 M Tris pH 8-1 .5 M NaCI, and incubated in 20X SSC for 5 minutes.
  • the DNA was transferred to a NYTRAN® Supercharge membrane.
  • the DNA was UV crosslinked to the membrane using a STRATALINKERTM UV crosslinker and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb.
  • a probe hybridizing to the 3' cbhll gene flanking sequence was generated using a PCR Dig Probe Synthesis Kit according to the manufacturer's instructions with the forward and reverse primers indicated below.
  • the PCR reaction was composed of 1X HERCULASE® Reaction Buffer, 400 nM each primer, 200 ⁇ DIG-labeled dUTP-containing dNTPs, 150 ng of T. reesei RutC30 genomic DNA, and 1 .5 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase.
  • reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 51 °C for 30 seconds, and 72°C for 40 seconds; and 1 cycle at 72°C for 7 minutes.
  • EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 51 °C for 30 seconds, and 72°C for 40 seconds; and 1 cycle at 72°C for 7 minutes.
  • the probe was purified by 1 % agarose gel electrophoresis using TAE buffer where a 0.5 kb band corresponding to the probe was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42°C for approximately 17 hours. The membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C.
  • probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that the three transformants contained the replacement cassette at the cbhll locus and all three (designated JfyS 139/144-5, -6, and -10) were chosen for curing the hpt and tk markers.
  • a fresh plate of spores for each transformant was generated by transferring a plug of a 7 day old culture grown on a PDA plate at 28°C to a new PDA plate and incubating for 7 days at 28°C. Spores were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemacytometer and 10 5 and 10 4 spores were spread onto 150 mm plates containing TrMM-G medium supplemented with 1 ⁇ FdU.
  • FdU-resistant spore isolates for each transformant were obtained from the plate containing 10 5 spores and approximately 100 FdU-resistant spore isolates for each transformant from the plate containing 10 4 spores. Eight spore isolates were picked for strains JfyS139/144-5 and -6 and four were picked for strain JfyS139/144-10. Each isolate 1 to 8 from primary transformant 5 was designated JfyS 139/144-5A to -5H. Isolates 1 to 8 from primary transformant 6 were designated JfyS 139/144-6A to 6H. Isolates from primary transformant 10 were designated JfyS139/144-1 OA to 10D for isolates 1 to 4. Spore PCR was conducted as described above, using the forward and reverse primers shown below, to confirm the hpt and tk markers had been correctly excised.
  • the primers annealed to the 5' (forward) and 3' (reverse) flanking sequences used for the cbhll gene replacement.
  • Strains from which the hpt/tk cassette had been correctly excised would display a 3.5 kb fragment while those with the markers intact would display an 8 kb fragment.
  • the PCR screen indicated that all of the spore isolates had correctly excised the hpt/tk cassette.
  • T reesei ku70 gene upstream sequence (consisting of 989 bp from upstream of the ku 70 coding sequence and the first 1010 bp of the ku 70 coding sequence) was amplified from T reesei 981 -0-8 genomic DNA using the sequence-specific forward and reverse primers shown below (SEQ ID NOs: 57 and 58).
  • T reesei ku70 gene downstream sequence (consisting of a 500 bp segment repeated from the 3' end of the 1010 bp segment of the ku70 coding sequence amplified in the upstream PCR product, and a 1067 bp segment containing the remainder of the ku 70 coding sequence, and 461 bp from downstream of the ku 70 coding sequence) was amplified from T reesei 981 -0-8 genomic DNA using the sequence-specific forward and reverse primers shown below (SEQ ID NOs: 59 and 60). T reesei 981-0-8 genomic DNA was prepared according to the procedure described in Example 1.
  • Reverse primer 5'-ATCAGCCCCGAGACGGCGCCGCGTTTAAACAATTCGTAATCATGGTCATAGCTGT-3' (SEQ ID NO: 56)
  • the reaction was composed of 100 ng of T. reesei 981-0-8 genomic DNA, 200 ⁇ dNTPs, 1 ⁇ of each primer (SEQ ID NO: 55 and 56), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer (New England Biolabs, Inc., Ipswich, MA, USA), and 1 .0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA) in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes.
  • the PCR product was separated by 1 % agarose gel electrophoresis using TAE buffer where a 2.692 kb fragment was excised from the gels and extracted using a MINELUTE® Gel Extraction Kit.
  • the reactions were composed of 100 ng of pJfyS139-B, 200 ⁇ dNTPs, 1 ⁇ of each primer (SEQ ID NOs: 57 and 58 or 59 and 60, respectively), 1 X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes.
  • the PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where 1 .999 kb and 2.028 kb fragments were separately excised from the gels and extracted using a MINELUTE® Gel Extraction Kit.
  • the fourth DNA segment was generated from a restriction enzyme digestion of pJfyS139-B with Not I and Bam HI.
  • the reaction was composed of 5 ⁇ g of pJfyS139-B, 10 units of Not I, 20 units of Bam HI, and 20 ⁇ of Restriction Enzyme Buffer 2 (New England Biolabs, Inc., Ipswich, MA, USA) in a total volume of 50 ⁇ .
  • the reaction was incubated for 1 hour at 37°C and then separated by 1 % agarose gel electrophoresis using TAE buffer where a 4.400 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 50 ng of the Not ⁇ l Bam Hl-digested pJfyS139-B, 50 ng of the 1.999 kb ku 70 gene upstream PCR product, 50 ng of the 2.028 kb ku 70 gene downstream PCR product, 50 ng of the 2.692 kb ampicillin resistance marker and prokaryotic origin of replication PCR product, and 1 ⁇ of IN-FUSION® Enzyme in a 10 ⁇ reaction volume. The reaction was incubated for 15 minutes at 37°C followed by 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction.
  • E. coli XL10 GOLD® competent cells (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and then 500 ⁇ of NZY+ medium, pre-heated to 42°C, were added. The tubes were incubated at 37°C with shaking at 200 rpm for 40 minutes and then plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Hind III and Xba I and positive clones sequenced to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pTH239.
  • Example 8 Repair of the ku70 gene in the A. fumigatus cbhl and cbh2 replacement strain JfyS139/144-10B
  • T. reesei JfyS139/144-10B The native Trichoderma reesei ku70 gene was repaired in strain T. reesei JfyS139/144-10B (Example 6) in order to facilitate strain manipulation steps requiring the function of the ku 70 gene in non-homologous end-joining.
  • T. reesei JfyS129/144-10B was transformed with 23 x 2 of Pme l-linearized pTH239 (Example 7) according to the procedure described in Example 2. Nineteen transformants were obtained and each one was separately transferred to a PDA plate and incubated for 7 days at 28°C.
  • a set of PCR primers shown below were designed to amplify across the disrupted region of the ku70 coding sequence to distinguish between the host genome with the disruption in the ku70 coding sequence (848 bp) and the pTH239 targeted strain of interest (606 bp).
  • the PCR reaction was composed of 1X ADVANTAGE® Genomic LA Polymerase Reaction Buffer (Clontech, Mountain View, CA, USA), 400 nM of each primer, 200 ⁇ dNTPs, 1 ⁇ of microwaved TE-spore mixture (described above), and 1.0 unit of ADVANTAGE® Genomic LA Polymerase (Clontech, Mountain View, CA, USA).
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds; and 1 cycle at 72°C for 7 minutes.
  • All twenty-two spore isolates (#19A-V) were screened by PCR for excision of the hpt/tk marker region present between the homologous repeats of the ku 70 coding sequence within the repair cassette.
  • a sterile inoculating loop was used to collect spores from a 7 day old PDA plate. The spores were transferred to a tube containing 25 ⁇ of 1 mM EDTA-10 mM Tris buffer and microwaved on high for 1 minute. A 1 ⁇ aliquot of the spore mixture was added directly to the PCR reaction as template genomic DNA.
  • a set of PCR primers shown below were designed to amplify across the hpt/tk region to distinguish between the presence (6 kb) or absence (1 .1 kb) of the hpt/tk region.
  • the PCR reaction was composed of 1X ADVANTAGE® Genomic LA Polymerase Reaction Buffer, 400 nM of each primer (below), 200 ⁇ dNTPs, 1 ⁇ of microwaved TE-spore mixture (described above), and 1.0 unit of ADVANTAGE® Genomic LA Polymerase.
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 30 cycles each at 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 6 minutes; and 1 cycle at 72°C for 7 minutes.
  • Forward primer :
  • genomic DNA was digested with (1 ) 5 units and 10 units, respectively, of Asc I and Xho I or (2) 5 units and 25 units, respectively, of Asc I and Apa I in a 50 ⁇ reaction volume and subjected to 1 % agarose electrophoresis using TAE buffer.
  • the DNA in the gel was depurinated with one 10 minute wash in 0.25 N HCI, denatured with two 15 minute washes in 0.5 N NaOH-1.5 M NaCI, neutralized with one 30 minute wash in 1 M Tris pH 8-1 .5 M NaCI, and incubated in 20X SSC for 5 minutes.
  • the DNA was transferred to a NYTRAN® Supercharge membrane using a TURBOBLOTTERTM System according to the manufacturer's protocol.
  • the DNA was UV crosslinked to the membrane using a STRATALINKERTM UV Crosslinker and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb.
  • a probe hybridizing to the 3' end of the ku 70 coding sequence was generated using a
  • PCR Dig Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions with the forward and reverse primers shown below.
  • the PCR reaction was composed of 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, 1 ⁇ of each primer, 200 ⁇ dNTPs, 165 ng of T. reesei 981 -0-8 genomic DNA, and 1 .0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase.
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 35 cycles each at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 15 seconds; and 1 cycle at 72°C for 10 minutes.
  • the 0.5 kb probe template was purified by 1 % agarose gel electrophoresis using TAE buffer and excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
  • the purified PCR product was used to generate a DIG-labeled probe as specified by the manufacturer's instructions using the primers and amplification conditions specified above.
  • the 0.5 kb DIG-labeled probe was purified by 1 % agarose gel electrophoreseis using TAE buffer and excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution.
  • Hybridization was performed at 42°C for 15-17 hours. The membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C.
  • the probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that all spore isolates contained the repair/replacement cassette at the ku70 locus and were cured of the hpt and tk markers.
  • T. reesei 981-O-8.5#10B+Ku70#19L3 was chosen for further transformations.
  • Example 9 Construction of pDM286 expressing a Penicillium sp. GH61A polypeptide
  • Penicillium sp. (emersonii) GH61A polypeptide coding sequence (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced amino acid sequence]) was amplified from plasmid pGH61 D23Y4 (WO 201 1/041397) using the gene-specific forward and reverse primers shown below.
  • the region in italics represents vector homology to the site of insertion for an IN-FUSION® reaction.
  • the amplification reaction was composed of 30 ng of pGH61 D23Y4 DNA, 50 pmoles of each of the primers listed above, 1 ⁇ of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, 1X PHUSIONTM High-Fidelity Hot Start DNA Polymerase Buffer, and 1 unit of PHUSIONTM High-Fidelity Hot Start DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 35 cycles each at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle at 72°C for 10 minutes.
  • the PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where an approximately 0.9 kb fragment was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's protocol.
  • Plasmid pMJ09 (WO 2005/047499) was digested with Nco I and Pac I, isolated by 1.0% agarose gel electrophoresis in 1 mM disodium EDTA-50 mM Tris base-50 mM boric acid (TBE) buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • the 0.9 kb PCR product was inserted into the gel-purified Nco ⁇ IPac I digested pMJ09 using an IN-FUSIONTM Advantage PCR Cloning Kit according to the manufacturer's protocol.
  • the IN-FUSIONTM reaction was composed of 1X IN-FUSIONTM Reaction Buffer, 180 ng of the gel-purified Nco ⁇ IPac I digested pMJ09, 108 ng of the 0.9 kb PCR product, and 1 ⁇ of IN-FUSIONTM Enzyme in a 10 ⁇ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 ⁇ of TE were added to the reaction.
  • Plasmid pDM286 can be digested with Pme I to generate an approximately 5.4 kb fragment for T. reesei transformation.
  • the 5.4 kb fragment contains the expression cassette composed of the T. reesei Cel7A cellobiohydrolase I gene promoter, P. emersonii GH61A polypeptide coding sequence, and T. reesei Cel7A cellobiohydrolase I gene terminator.
  • the 5.4 kb fragment also contains the Aspergillus nidulans acetamidase ⁇ amdS) gene.
  • Example 10 Generation of a Trichoderma reesei expression vector encoding Aspergillus fumigatus beta-glucosidase (Cel3A) mutant gene
  • a variant of the Aspergillus fumigatus Family 3A beta-glucosidase containing the substitutions G142S, Q183R, H266Q, and D703G was constructed by performing site- directed mutagenesis on pEJG97 (WO 2005/074647) using a QUIKCHANGE® Multi Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA).
  • a summary of the oligos used for the site-directed mutagenesis are shown in Table 1.
  • the resulting variant plasmid pDFng128-6 was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, CA, USA).
  • the variant plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) to verify the changes.
  • Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-glucosidase mutant coding sequence from plasmid pDFng128-6.
  • An IN-FUSIONTM Cloning Kit was used to clone the fragment directly into the expression vector pMJ09. Bold letters represent coding sequence. The remaining sequence is homologous to insertion sites of pMJ09.
  • the amplification was performed in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 94°C for 2 minute; 30 cycles each at 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 1 minute; and a final elongation at 68°C for 7 minutes.
  • the heat block then went to a 4°C soak cycle.
  • the reaction products were isolated by 0.7% agarose gel electrophoresis in TBE buffer where an approximately 3.1 kb product band was observed on the gel.
  • the PCR reaction was purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • Plasmid pMJ09 was digested with Nco I and Pac I, isolated by 1 .0% agarose gel electrophoresis in TBE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • the 3.1 kb gene fragment and the digested vector were ligated together using an I NFUSIONTM Cloning Kit resulting in pDFng1 13-3 ( Figure 5) in which transcription of the beta- glucosidase mutant coding sequence was under the control of a promoter from the Trichoderma reesei cbhl gene.
  • the ligation reaction (20 ⁇ ) was composed of 1 X IN- FUSIONTM Buffer, 1X BSA, 1 ⁇ of IN-FUSIONTM Enzyme (diluted 1 :10), 200 ng of the gel- purified Nco ⁇ IPac I digested pMJ09, and 172.2 ng of the purified 3.1 kb PCR product.
  • the reaction was incubated at 37°C for 15 minutes followed by 50°C for 15 minutes.
  • Two ⁇ of the reaction was used to transform £ coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, CA, USA).
  • the £ coli transformation reactions were spread onto 2XYT plus ampicillin plates.
  • An £. coli transformant containing pDFng133-3 was prepared using a BIOROBOT® 9600.
  • the Aspergillus fumigatus beta-glucosidase mutant insert in pDFng133 was confirmed by DNA sequencing.
  • the Humicola insolens endoglucanase V full-length coding region was PCR amplified from pMJ05 (US 2004/0248258 A1 ) as template with the primers shown below.
  • the underlined portions are Sph I and a Hind III sites introduced by the Car- F2 sense primer.
  • the bold portion is an Eco Rl site introduced by the Car-R2 antisense primer.
  • the amplification reactions (50 ⁇ ) were composed of 1X ThermoPol Reaction Buffer (New England Biolabs, Inc., Ipswich, MA USA), 0.3 mM dNTPs, 10 ng of pMJ05 DNA, 0.3 ⁇ Car-F2 sense primer, 0.3 ⁇ Car-R2 antisense primer, and 2.5 units of VENT® DNA polymerase (New England Biolabs, Inc., Ipswich, MA USA).
  • the reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 30 cycles each at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds (15 minute final extension).
  • reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 900 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • the 900 bp PCR fragment was then digested with Eco Rl and Hind III and subjected to a QIAQUICK® PCR Purification Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol.
  • Plasmid pMJ05 was digested with Eco Rl and Hind III, isolated by 0.7% agarose gel electrophoresis in TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • the 900 bp Eco Rl and Hind III digested PCR fragment was ligated using T4 DNA ligase (Roche, Indianapolis, IN, USA) into Eco Rl and Hind III digested pMJ05.
  • the ligation reaction was composed of 50 ng of the Eco Rl and Hind III digested pMJ05, 33 ng of the Eco Rl and Hind III digested 0.9 kb PCR fragment, 1X Ligase Buffer (Roche, Indianapolis, IN, USA), and 2 units of T4 DNA ligase in a final volume of 20 ⁇ .
  • the reaction was incubated at 15°C for 17 hours and 2 ⁇ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol.
  • the cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Sph I and Bam HI to determine the presence and orientation of the insert and positive clones were sequenced.
  • a clone containing the Humicola insolens endoglucanase V coding region with no PCR errors was designated pSMai139 ( Figure 6).
  • Plasmid pSMai143 was constructed by amplifying 620 bp of the Trichoderma reesei cellobiohydrolase Cel6A promoter from Trichoderma reesei RutC30 genomic DNA using primers 994148 and 994149 shown below.
  • the underlined portion is a Sal I site introduced by primer 994148.
  • the bold portion is a "CAT" sequence introduced by primer 994149.
  • the amplification reactions (50 ⁇ ) were composed of 1X ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 0.3 ⁇ 994148 sense primer, 0.3 ⁇ 994149 antisense primer, and 2.5 units of Vent DNA polymerase.
  • the reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 30 cycles each at 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds (15 minute final extension).
  • reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 620 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • Plasmid pSMai139 was digested with Sph I, 3'-protruding end blunted with T4 DNA polymerase and then digested with Sal I.
  • the digested DNA was isolated by 0.7% agarose gel electrophoresis in TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • the 620 bp Sal I digested PCR fragment was ligated using T4 DNA ligase into Sph I and Sal I digested pSMai139.
  • the ligation reaction was composed of 50 ng of the Sph I and Sal I digested pSMai139, 22 ng of the Sal I digested 0.62 kb PCR fragment, "IX Ligase Buffer, and 2 units of T4 DNA ligase in a final volume of 20 ⁇ .
  • the reaction was incubated at 15°C for 17 hours and 2 ⁇ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 ⁇ of SOC medium were added.
  • the tubes were incubated at 37°C, 200 rpm for 1 hour and 250 ⁇ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.
  • the resulting transformants were screened by restriction digestion analysis with Eco Rl to determine the presence and orientation of the insert and positive clones were sequenced.
  • a clone containing the Trichoderma reesei cellobiohydrolase Cel6A promoter with no PCR errors was designated pSMai143 ( Figure 7).
  • Expression vector pAG121 with an Nco I restriction site was constructed by performing site-directed mutagenesis on pSMai143 (Example 12) using a QUIKCHANGE® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using the primers shown below. The mutagenesis was performed according to manufacturer's recommendations using 20 ng of plasmid pAG121 and 12.5 ⁇ primers in a final volume of 50 ⁇ .
  • the resulting variant plasmid pAG121 was prepared using a BIOROBOT® 9600.
  • the variant plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer to verify the changes.
  • Example 14 Construction of a Trichoderma reesei expression vector, pSMai229, encoding an Aspergillus fumigatus beta-glucosidase (Cel3A) mutant gene
  • the Aspergillus fumigatus beta-glucosidase (Cel3A) mutant coding sequence was PCR amplified from pDFng133-3 using primers 061 1689 and 061 1690 shown below. The regions in bold represent pAG121 vector homology to the site of insertion for IN-FUSION® cloning.
  • the amplification reaction was composed of 25 ng of pDFng133-3 DNA, 200 ⁇ dNTP's, 0.4 ⁇ primers, 1X PHUSION® Buffer, and 1 unit of PHUSION® Hot Start High Fidelity DNA Polymerase in a final volume of 50 ⁇ .
  • the amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 30 seconds, 56°C for 30 seconds, and 72°C for 3 minutes 30 seconds; and 1 cycle at 72°C for 15 minutes.
  • PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 3100 bp fragment was excised from the gel and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions. The fragment was then cloned to the largest fragment of pAG121 digested with Nco ⁇ and Spel using an IN-FUSIONTM Advantage PCR Cloning Kit resulting in pSMai229 ( Figure 8).
  • the ligation reaction (10 ⁇ ) was composed of 1X IN-FUSIONTM Buffer, 1 ⁇ of IN-FUSIONTM Enzyme, 100 ng of pAG121 digested with Nco I and Spe I, and 142 ng of 3100 bp purified PCR product.
  • the reaction was incubated at 37°C for 15 minutes followed by 15 minutes at 50°C. After diluting the reaction mix with 50 ⁇ of TE buffer (pH 8), 2.5 ⁇ of the reaction was used to transform £. coli ONE SHOT® TOP10 competent cells according to the manufacturer's protocol.
  • An £. coli transformant containing pSMai229 was detected by restriction digestion and plasmid DNA was prepared using a BIOROBOT® 9600.
  • the Aspergillus fumigatus beta-glucosidase (Cel3A) mutant insert in pSMai229 was confirmed by DNA sequencing.
  • Example 15 Co-transformation of pDM286 and pSMai229 into Trichoderma reesei 981 - O-8.5#10B+Ku70#19L3
  • pDM286 and pSMai229 were digested with Pme I. Each digestion reaction was purified by 1 % agarose gel electrophoresis in TAE buffer, a DNA band was excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit. Transformation was performed by adding 0.7-1.7 ⁇ g of Pme I digested and gel-purified pSMai229 and 0.7-2.0 of pDM286 to 100 ⁇ of Trichoderma reesei T. reesei 981 -0- 8#10B+Ku70#19L3 protoplast solution and mixed gently. PEG buffer (250 ⁇ ) was added, mixed, and incubated at 34°C for 30 minutes.
  • the supernatants were assayed for beta-glucosidase activity using p-nitrophenyl- beta-D-glucopyranoside as substrate. Briefly, culture supernatants were diluted appropriately in 0.1 M succinate-0.01 % TRITON® X-100 pH 5.0 buffer (sample buffer) followed by a series dilution from 0-fold to 1/3-fold to 1/9-fold of the diluted sample. T. reesei RutC30 fermentation broth was initially diluted 1/64 followed with 2-fold dilution steps down to a 16- fold dilution in the sample buffer to establish the assay linear range. A total of 20 ⁇ of each dilution was transferred to a 96-well flat bottom plate.
  • the Aspergillus fumigatus strain NN051616 GH3 beta-xylosidase SEQ ID NO: 15
  • Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-xylosidase gene from genomic DNA. Genomic DNA was prepared as described in Example 1 . An I N-FUSIONTM Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pAILo2 (WO 2005/074647), without the need for restriction digests and ligation.
  • Bold letters represent coding sequence. The remaining sequence is homologous to the insertion sites of pAILo2.
  • the amplification was performed using an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 94°C for 2 minutes; 10 cycles each at 94°C for 15 seconds, 56.5°C for 30 seconds, and 72°C for 2 minutes; and 20 cycles each at 94°C for 15 seconds, 56.5°C for 30 seconds, and 72°C for 2 minutes plus 5 seconds per successive cycle.
  • the heat block was then held at 72°C for 7 minutes followed by a 4°C soak cycle.
  • reaction products were isolated by 1 .0% agarose gel electrophoresis using TAE buffer where a 2.4 kb product band was excised from the gel and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions.
  • the fragment was then cloned into pAILo2 using an IN-FUSIONTM Advantage PCR Cloning Kit.
  • the vector was digested with Nco I and Pac I .
  • the fragment was purified by 1 % agarose gel electrophoresis using TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit.
  • the gene fragment and the digested vector were combined together in a reaction resulting in the expression plasmid pAG57, in which transcription of the Aspergillus fumigatus beta-xylosidase coding sequence was under the control of the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase).
  • the reaction (20 ⁇ ) was composed of 1X IN-FUSIONTM Buffer, 1X BSA, 1 ⁇ of IN-FUSIONTM Enzyme (diluted 1 :10), 182 ng of pAILo2 digested with Nco I and Pac I, and 97.7 ng of the Aspergillus fumigatus beta-xylosidase purified PCR product.
  • the reaction was incubated at 37°C for 15 minutes followed by 15 minutes at 50°C.
  • the reaction was diluted with 40 ⁇ of TE buffer and 2.5 ⁇ of the diluted reaction were used to transform E. coli TOP10 Competent cells. An £.
  • coli transformant containing pAG57 ( Figure 9) was identified by restriction enzyme digestion and plasmid DNA was prepared using a BIOROBOT® 9600.
  • the pAG57 plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer to verify the sequence.
  • Example 17 Construction of pDFng 124-1 expressing an Aspergillus fumigatus beta- xylosidase
  • Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-xylosidase from pAG57 (Example 16).
  • An IN-FUSIONTM Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pMJ09, without the need for restriction digestion and ligation.
  • Bold letters represent coding sequence. The remaining sequence was homologous to insertion sites of pMJ09.
  • reaction products were isolated by 0.7% agarose gel electrophoresis in TBE buffer where a 2.4 kb product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • Plasmid pMJ09 was digested with Nco I and Pac I , isolated by 0.7% agarose gel electrophoresis in TBE buffer, and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The gene fragment and the digested vector were ligated together using an INFUSIONTM Advantage PCR Cloning Kit resulting in pDFng124-1 ( Figure 10) in which transcription of the beta-xylosidase coding sequence was under the control of the Trichoderma reesei cbhl gene promoter.
  • the ligation reaction (20 ⁇ ) was composed of 1X IN-FUSIONTM Buffer, 1 ⁇ of IN-FUSIONTM Enzyme (diluted 1 :10), 200 ng of pMJ09 digested with Nco I and Pac I, and 100 ng of the purified beta-xylosidase PCR product.
  • the reaction was incubated at 37°C for 15 minutes followed by 50°C for 15 minutes.
  • Two ⁇ of the reaction were used to transform E. coli XL10 SOLOPACK® Gold Supercompetent cells according to manufacturer's instructions.
  • An E. coli transformant containing pDFng124-1 was prepared using a BIOROBOT® 9600. The Aspergillus fumigatus beta-xylosidase insert in pDFng124-1 was confirmed by DNA sequencing.
  • Example 18 Construction pSaMe-AFGH10 expressing an Aspergillus fumigatus xylanase
  • Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus GH10 xylanase from pHyGe001 (WO 2006/078256).
  • An INFUSIONTM Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pMJ09, without the need for restriction digestion and ligation.
  • Bold letters represent coding sequence. The remaining sequence was homologous to the insertion sites of pMJ09.
  • An EPPENDORF® MASTERCYCLER® 5333 epgradient S was used to amplify the DNA fragment programmed for 1 cycle at 95°C for 2 minutes; and 30 cycles each at 94°C for 15 seconds, 55°C for 30 seconds, and 68°C for 1 minute. After the 30 cycles, the reaction was incubated at 72°C for 10 minutes and then cooled to 4°C until further processing.
  • reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 1.4 kb product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
  • Plasmid pMJ09 was digested with Nco I and Pac I and purified by agarose gel electrophoresis as described above. The gene fragment and the digested vector were ligated together in a reaction resulting in the expression plasmid pSaMe-AfGH10 in which transcription of the xylanase coding sequence was under the control of the T. reesei cbhl gene promoter.
  • the ligation reaction (50 ⁇ ) was composed of 1X IN-FUSIONTM Buffer, 1X BSA, 1 ⁇ of IN-FUSIONTM enzyme (diluted 1 :10), 100 ng of pMJ09 digested with Nco I and Pac I, and 100 ng of the Aspergillus fumigatus xylanase purified PCR product.
  • the reaction was incubated at room temperature for 30 minutes.
  • One ⁇ of the reaction was used to transform E. coli XL10 SOLOPACK® Gold cells.
  • An E. coli transformant containing pSaMe- AfGH10 ( Figure 1 1 ) was detected by restriction enzyme digestion and plasmid DNA was prepared using a BIOROBOT® 9600.
  • pSaMe-AFGH10 and pDFng124-1 were digested with Pme I. Each digestion reaction was purified by 0.65% agarose gel electrophoresis in TAE buffer, a DNA band was excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit. Transformation was performed by adding 2 ⁇ g of Pme I digested and gel-purified pDFng124- 1 and 1.72 ⁇ g of pSaMe-AfGH10 to 100 ⁇ of Trichoderma reesei strain RutC30 protoplast solution and mixed gently. PEG buffer (250 ⁇ ) was added, mixed, and incubated at 34°C for 30 minutes.
  • strains were selected based on high expression of beta-xylosidase and xylanase polypeptide and were spore purified by adding spores collected on a 10 ⁇ inoculation loop to 1.5 ml of 0.01 % TWEEN® 20. Spore dilutions of 1 :1500 and 1 :150 were spread onto 150 mm COVE plates and cultured for 4 days at 28°C. Four spore isolates per strain (total of 16 isolates) were obtained and transferred to COVE2 + 10 mM uridine plates and cultivated at 28°C for 9 days. The shake flask and SDS-PAGE procedures were repeated for the first round spore isolates.
  • Eight strains were selected based on high expression of beta-xylosidase and xylanase polypeptide and were spore purified a second time as described above resulting in four spore isolates per strain (total of 32 isolates). The shake flask and SDS-PAGE procedures were repeated for the second round spore isolates. The final strain was selected based on high expression of beta-xylosidase and xylanase polypeptide and designated 06HY4.
  • Corn stover was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1 .4 wt % sulfuric acid at 165°C and 107 psi for 8 minutes.
  • the water-insoluble solids in the pretreated corn stover (PCS) contained 56.5% cellulose, 4.6% hemicelluloses, and 28.4% lignin.
  • Cellulose and hemicellulose were determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003.
  • Milled unwashed PCS was prepared by milling whole slurry PCS in a Cosmos ICMG
  • the hydrolysis of PCS was conducted using 2.2 ml deep-well plates (Axygen, Union City, CA, USA) in a total reaction volume of 1 .0 ml.
  • the hydrolysis was performed with 50 mg of insoluble PCS solids per ml of 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate and various protein loadings of various enzyme compositions (expressed as mg protein per gram of cellulose).
  • Enzyme compositions were prepared and then added simultaneously to all wells in a volume ranging from 50 ⁇ to 200 ⁇ , for a final volume of 1 ml in each reaction.
  • the plates were then sealed using an ALPS-300TM plate heat sealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and incubated at a specific temperature for 72 hours. All experiments reported were performed in triplicate.
  • the sugar concentrations of samples diluted in 0.005 M H 2 S0 4 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.05% w/w benzoic acid-0.005 M H 2 S0 4 at 65°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION®, AGI LENT® 1 100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. The resultant glucose and cellobiose equivalents were used to calculate the percentage of cellulose conversion for each reaction.
  • AMINEX® HPX-87H column Bio-Rad Laboratories, Inc., Hercules, CA, USA
  • Glucose, cellobiose, and xylose were measured individually. Measured sugar concentrations were adjusted for the appropriate dilution factor. In case of unwashed PCS, the net concentrations of enzymatically-produced sugars were determined by adjusting the measured sugar concentrations for corresponding background sugar concentrations in unwashed PCS at zero time points. All HPLC data processing was performed using MICROSOFT EXCELTM software (Microsoft, Richland, WA, USA).
  • An enzyme composition comprising an Aspergillus fumigatus cellobiohydrolase I; an Aspergillus fumigatus cellobiohydrolase II; an Aspergillus fumigatus beta-glucosidase variant; a Penicillium sp.
  • GH61 polypeptide having cellulolytic enhancing activity, an Aspergillus fumigatus xylanase, and an Aspergillus fumigatus beta-xylosidase (designated “enzyme composition #1 ") was compared to an enzyme composition comprising a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) (designated "enzyme composition #2").
  • a Protein Loading (mg EP/g cellulose) versus Percent Converison (%) graph was generated. Using linear interpolation the protein loading required to reach a certain percent conversion can be determined. In this case, 80% conversion of glucan to glucose equivalents was chosen to determine relative improvements of enzyme composition 1 as compared to enzyme composition 2.
  • the results of this assay as shown in Figure 12 indicated that enzyme composition 1 is able to reach 80% conversion with 4.1 mg EP/g cellulose, whereas enzyme compositoni 2 is able to reach the same conversion target with 7.3 mg EP/g cellulose. This represents a 1.78 fold improvement in performance per milligram of protein for enzyme composition 1 over enzyme composition 2 or a 1.78 fold reduction in protein requirement to reach 80% conversion.
  • An enzyme composition comprising: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the enzyme composition of paragraph 1 wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2; (iii) a cellobiohydrolase I encoded by a polynucle
  • the enzyme composition of paragraph 1 wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4; (iii) a cellobiohydrolase II encoded by a polynucle
  • the enzyme composition of paragraph 1 wherein the Aspergillus fumigatus beta- glucosidase or homolog thereof is selected from the group consisting of: (i) a beta- glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta- glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g.
  • beta-glucosidase variant comprises a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 6, wherein the variant has beta- glucosidase activity.
  • the parent beta-glucosidase of the variant is (a) a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 6; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or (e) a fragment of the mature polypeptide of SEQ ID NO: 6, which has beta-
  • the enzyme composition of any of paragraphs 5-12, wherein the variant comprises the substitutions G142S and Q183R; G142S and H266Q; G142S and D703G; Q183R and H266Q; Q183R and D703G; H266Q and D703G; G142S, Q183R, and H266Q; G142S, Q183R, and D703G; G142S, H266Q, and D703G; Q183R, H266Q, and D703G; or G142S, Q183R, H266Q, and D703G.
  • the enzyme composition of any of paragraphs 1-14 which further comprises one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase or homolog thereof, (ii) an Aspergillus fumigatus beta-xylosidase or homolog thereof; or (iii) a combination of (i) and (ii).
  • the enzyme composition of any of paragraphs 1-17 which further comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
  • a recombinant filamentous fungal host cell comprising polynucleotides encoding: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • Aspergillus fumigatus beta-glucosidase or homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6; (iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of
  • beta- glucosidase variant comprises a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 6, wherein the variant has beta-glucosidase activity.
  • the parent beta-glucosidase of the variant is (a) a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 6; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or (e) a fragment of the mature polypeptide of S
  • Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of: (i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 8; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii) a GH61 polypeptide having cellul
  • the recombinant filamentous fungal host cell of any of paragraphs 23-36 which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase; (ii) an Aspergillus fumigatus beta-xylosidase; and (iii) a combination of (i) and (ii).
  • Trichoderma cell selected from the group consisting of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.
  • Trichoderma reesei Trichoderma reesei.
  • cellobiohydrolase I gene encodes a cellobiohydrolase I selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 18; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 18; (iii) a cellobiohydrolase I encoded by
  • cellobiohydrolase II encodes a cellobiohydrolase II selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 20; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 20; (iii) a cellobiohydrolase II encoded by
  • beta-glucosidase gene encodes a beta-glucosidase selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 22; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 22; (iii) a beta-glucos
  • xylanase I encodes a xylanase I selected from the group consisting of: (i) a xylanase I comprising or consisting of the mature polypeptide of SEQ ID NO: 24; (ii) a xylanase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 24; (iii) a xylanase I selected from the group consisting of: (i) a xylanase
  • xylanase II encodes a xylanase II selected from the group consisting of: (i) a xylanase II comprising or consisting of the mature polypeptide of SEQ ID NO: 26; (ii) a xylanase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 26; (iii) a xylanase II selected from the group consisting of: (i) a xylanase
  • xylanase III encodes a xylanase III selected from the group consisting of: (i) a xylanase III comprising or consisting of the mature polypeptide of SEQ ID NO: 28; (ii) a xylanase III comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 28; (iii) a xylanase III selected from the group consisting of: (i) a xylanase
  • beta- xylosidase gene encodes a beta-xylosidase selected from the group consisting of: (i) a beta- xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 30 (ii) a beta- xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g.
  • the recombinant filamentous fungal host cell of any of paragraphs 23-55 which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
  • a method of producing an enzyme composition comprising: (a) cultivating the host cell of any of paragraphs 23-64 under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
  • a process for degrading a cellulosic material comprising: treating the cellulosic material with the enzyme composition of any of paragraphs 1-22.
  • a process for producing a fermentation product comprising: (a) saccharifying a cellulosic material with the enzyme composition of any of paragraphs 1-22; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • a process of fermenting a cellulosic material comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with the enzyme composition of any of paragraphs 1 -22.
  • Trichoderma endoglucanase I is a Trichoderma reesei endoglucanase I.
  • Trichoderma endoglucanase II is a Trichoderma reesei endoglucanase II.

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Abstract

The present invention relates to recombinant filamentous fungal host cells producing cellulolytic enzyme compositions and methods of producing and using the compositions.

Description

CELLULOLYTIC ENZYME COMPOSITIONS AND USES THEREOF
Statement as to Rights to Inventions Made Under Federally Sponsored Research and Development
This invention was made with Government support under Cooperative Agreement DE-FC36-08GO18080 awarded by the Department of Energy. The government has certain rights in this invention.
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Background of the Invention
Field of the Invention
The present invention relates to cellulolytic enzyme compositions; recombinant filamentous fungal host cells producing the cellulolytic enzyme compositions and methods of producing and using the compositions.
Description of the Related Art
Cellulose is a polymer of glucose linked by beta-1 ,4-bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta- glucosidases hydrolyze cellobiose to glucose.
The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.
WO 201 1/057140 discloses an Aspergillus fumigatus cellobiohydrolase I and gene thereof. WO 201 1/057140 discloses an Aspergillus fumigatus cellobiohydrolase II and gene thereof. WO 2005/047499 discloses an Aspergillus fumigatus beta-glucosidase and gene thereof. WO 2006/078256 discloses Aspergillus fumigatus GH10 xylanases. WO 201 1/057140 discloses an Aspergillus fumigatus beta-xylosidase and gene thereof. WO 201 1/041397 discloses a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity and gene thereof.
There is a need in the art for new cellulolytic enzyme compositions that can deconstruct cellulosic material more efficiently.
The present invention provides cellulolytic enzyme compositions and methods of producing and using the compositions.
Summary of the Invention
The present invention relates to enzyme compositions, comprising (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
The present invention also relates to recombinant filamentous fungal host cells, comprising polynucleotides encoding (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
The present invention also relates to methods of producing an enzyme composition, comprising: (a) cultivating a filamentous fungal host cell of the present invention under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
The present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition of the present invention.
The present invention also relates to processes for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
The present invention further relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition of the present invention. Brief Description of the Figures
Figure 1 shows a restriction map of plasmid pJfyS139.
Figure 2 shows a restriction map of plasmid pJfyS142.
Figure 3 shows a restriction map of plasmid pJfyS144.
Figure 4 shows a restriction map of plasmid pDM286.
Figure 5 shows a restriction map of plasmid pDFng1 13-3.
Figure 6 shows a restriction map of plasmid pSMai139.
Figure 7 shows a restriction map of plasmid pSMai143.
Figure 8 shows a restriction map of plasmid pSMai229.
Figure 9 shows a restriction map of plasmid pAG57.
Figure 10 shows a restriction map of plasmid pDFng124-1.
Figure 1 1 shows a restriction map of plasmid pSaMe-AFGH10.
Figure 12 shows a comparison of percent conversion of pretreated corn stover (PCS) by an enzyme composition comprising an Aspergillus fumigatus cellobiohydrolase I; an Aspergillus fumigatus cellobiohydrolase II; an Aspergillus fumigatus beta-glucosidase variant; a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity, an Aspergillus fumigatus xylanase, and an Aspergillus fumigatus beta-xylosidase ("enzyme composition #1 ") to an enzyme composition comprising a blend of an Aspergillus aculeatus GH10 xylanase and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide ("enzyme composition #2").
Definitions
Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of the present invention, acetylxylan esterase activity is determined using 0.5 mM p- nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01 % TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 μηηοΐβ of p-nitrophenolate anion per minute at pH 5, 25°C.
Allelic variant: The term "allelic variant" means any of two or more (e.g., several) alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L- arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L- arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L- arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 μΙ for 30 minutes at 40°C followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D- glucosiduronate glucuronohydrolase (EC 3.2.1 .139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 μηηοΐβ of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40°C.
Aspartic protease: The term "aspartic protease" means a protease that uses an aspartate residue(s) for catalyzing the hydrolysis of peptide bonds in peptides and proteins. Aspartic proteases are a family of protease enzymes that use an aspartate residue for catalytic hydrolysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH (Szecsi, 1992, Scand. J. Clin. Lab. In vest. Suppl. 210: 5-22). For purposes of the present invention, aspartic protease activity is determined according to the procedure described by Aikawa et al., 2001 , J. Biochem. 129: 791 -794.
Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1 .21 ) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1 .0 μηιοΐΘ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p- nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20 (polyoxyethylene sorbitan monolaurate).
Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1 .37) that catalyzes the exo-hydrolysis of short beta^(4) xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. For purposes of the present invention, one unit of beta-xylosidase is defined as 1 .0 μηηοΐβ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D- xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20.
cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Cellobiohydrolase: The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1 .176) that catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described by Lever et al. , 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al. , 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283- 288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581 . In the present invention, the Tomme et al. method can be used to determine cellobiohydrolase activity.
Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1 ) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).
For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnS04, 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMI NEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-1 18, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix. In a preferred aspect, the cellulosic material is any biomass material. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.
In one aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue).
In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switchgrass. In another aspect, the cellulosic material is wheat straw.
In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
In another aspect, the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.
In another aspect, the cellulosic material is an aquatic biomass. As used herein the term "aquatic biomass" means biomass produced in an aquatic environment by a photosynthesis process. The aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.
The cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.
Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Endoglucanase: The term "endoglucanase" means an endo-1 ,4-(1 ,3;1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.
Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61 " or "GH61 " means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695- 696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
Feruloyl esterase: The term "feruloyl esterase" means a 4-hydroxy-3- methoxycinnamoyl-sugar hydrolase (EC 3.1 .1.73) that catalyzes the hydrolysis of 4-hydroxy- 3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 μηηοΐβ of p-nitrophenolate anion per minute at pH 5, 25°C.
Flanking: The term "flanking" means DNA sequences extending on either side of a specific DNA sequence, locus, or gene. The flanking DNA is immediately adjacent to another DNA sequence, locus, or gene that is to be integrated into the genome of a filamentous fungal cell.
Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide main; wherein the fragment has enzyme activity. In one aspect, a fragment contains at least 85%, e.g., at least 90% or at least 95% of the amino acid residues of the mature polypeptide of an enzyme.
Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.
High stringency conditions: The term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
Homologous 3' or 5' region: The term "homologous 3' region" means a fragment of DNA that is identical in sequence or has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a region in the genome and when combined with a homologous 5' region can target integration of a piece of DNA to a specific site in the genome by homologous recombination. The term "homologous 5' region" means a fragment of DNA that is identical in sequence to a region in the genome and when combined with a homologous 3' region can target integration of a piece of DNA to a specific site in the genome by homologous recombination. The homologous 5' and 3' regions must be linked in the genome which means they are on the same chromosome and within at least 200 kb of one another.
Homologous flanking region: The term "homologous flanking region" means a fragment of DNA that is identical or has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a region in the genome and is located immediately upstream or downstream of a specific site in the genome into which extracellular DNA is targeted for integration.
Homologous repeat: The term "homologous repeat" means a fragment of DNA that is repeated at least twice in the recombinant DNA introduced into a host cell and which can facilitate the loss of the DNA, i.e., selectable marker that is inserted between two homologous repeats, by homologous recombination. A homologous repeat is also known as a direct repeat.
Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non- naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
Low stringency conditions: The term "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide of an A. fumigatus cellobiohydrolase I is amino acids 27 to 532 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6) that predicts amino acids 1 to 26 of SEQ ID NO: 2 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus cellobiohydrolase II is amino acids 20 to 454 of SEQ ID NO: 4 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 4 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus beta-glucosidase is amino acids 20 to 863 of SEQ ID NO: 6 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 6 are a signal peptide. In another aspect, the mature polypeptide of a Penicillium sp. GH61 polypeptide is amino acids 26 to 253 of SEQ ID NO: 8 based on the SignalP program that predicts amino acids 1 to 25 of SEQ ID NO: 8 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus xylanase I is amino acids 18 to 364 of SEQ ID NO: 10 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 10 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus xylanase II is amino acids 20 to 323 of SEQ ID NO: 12 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 12 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus xylanase III is amino acids 20 to 397 of SEQ ID NO: 14 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 14 are a signal peptide. In another aspect, the mature polypeptide of an A. fumigatus beta-xylosidase is amino acids 21 to 792 of SEQ ID NO: 16 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 16 are a signal peptide.
In another aspect, the mature polypeptide of a T. reesei cellobiohydrolase I is amino acids 18 to 514 of SEQ ID NO: 18 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 18 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei cellobiohydrolase II is amino acids 19 to 471 of SEQ ID NO: 20 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 20 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei beta-glucosidase is amino acids 20 to 744 of SEQ ID NO: 22 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 22 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei xylanase I is amino acids 20 to 229 of SEQ ID NO: 24 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 24 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei xylanase II is amino acids 20 to 223 of SEQ ID NO: 26 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 26 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei xylanase III is amino acids 17 to 347 of SEQ ID NO: 28 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 28 are a signal peptide. In another aspect, the mature polypeptide of a T. reesei beta-xylosidase is amino acids 21 to 797 of SEQ ID NO: 30 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having enzyme activity. In one aspect, the mature polypeptide coding sequence of an A. fumigatus cellobiohydrolase I is nucleotides 79 to 1596 of SEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP program (Nielsen et al. , 1997, supra) that predicts nucleotides 1 to 78 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus cellobiohydrolase II is nucleotides 58 to 1700 of SEQ ID NO: 3 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 3 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus beta-glucosidase is nucleotides 58 to 2580 of SEQ ID NO: 5 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 5 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a Penicillium sp. GH61 polypeptide is nucleotides 76 to 832 of SEQ ID NO: 7 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 7 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus xylanase I is nucleotides 52 to 1 145 of SEQ ID NO: 9 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 9 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus xylanase II is nucleotides 58 to 1400 of SEQ ID NO: 1 1 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 1 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus xylanase III is nucleotides 107 to 1415 of SEQ ID NO: 13 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 106 of SEQ ID NO: 13 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of an A. fumigatus beta-xylosidase is nucleotides 61 to 2373 of SEQ ID NO: 15 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 15 encode a signal peptide.
In another aspect, the mature polypeptide coding sequence of a T. reesei cellobiohydrolase I is nucleotides 52 to 1545 of SEQ ID NO: 17 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 17 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei cellobiohydrolase II is nucleotides 55 to 1608 of SEQ ID NO: 19 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 19 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei beta-glucosidase is nucleotides 58 to 2612 of SEQ ID NO: 21 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 21 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei xylanase I is nucleotides 58 to 749 of SEQ ID NO: 23 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 23 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei xylanase II is nucleotides 58 to 778 of SEQ ID NO: 25 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 25 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei xylanase III is nucleotides 49 to 1349 of SEQ ID NO: 27 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 27 encode a signal peptide. In another aspect, the mature polypeptide coding sequence of a T. reesei beta-xylosidase is nucleotides 61 to 2391 of SEQ I D NO: 29 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 29 encode a signal peptide.
Medium stringency conditions: The term "medium stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
Medium-high stringency conditions: The term "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more (e.g., several) control sequences.
Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Polypeptide having cellulolytic enhancing activity: The term "polypeptide having cellulolytic enhancing activity" means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1 -50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvaerd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.
The GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20- fold.
Pretreated corn stover: The term "PCS" or "Pretreated Corn Stover" means a cellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, or neutral pretreatment.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having enzyme activity. In one aspect, a subsequence contains at least 85%, e.g., at least 90% or at least 95% of the nucleotides of the mature polypeptide coding sequence of an enzyme.
Subtilisin-like serine protease: The term "subtilisin-like serine protease" means a protease with a substrate specificity similar to subtilisin that uses a serine residue for catalyzing the hydrolysis of peptide bonds in peptides and proteins. Subtilisin-like proteases (subtilases) are serine proteases characterized by a catalytic triad of the three amino acids aspartate, histidine, and serine. The arrangement of these catalytic residues is shared with the prototypical subtilisin from Bacillus licheniformis (Siezen and Leunissen, 1997, Protein Science 6: 501-523). Subtilisin-like serine protease activity can be determined using a synthetic substrate, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) (Bachem AG, Bubendorf, Switzerland) in 100 mM NaCI-100 mM MOPS pH 7.0 at 50°C for 3 hours and then the absorbance at 405 nm is measured.
Targeted integration: The term "targeted integration" means the stable integration of extracellular DNA at a defined genomic locus.
Transformant: The term "transformant" means a cell which has taken up extracellular DNA (foreign, artificial or modified) and expresses the gene(s) contained therein.
Transformation: The term "transformation" means the introduction of extracellular DNA into a cell, i.e., the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
Trypsin-like serine protease: The term "trypsin-like serine protease" means a protease with a substrate specificity similar to trypsin that uses a serine residue for catalyzing the hydrolysis of peptide bonds in peptides and proteins. For purposes of the present invention, trypsin-like serine protease activity is determined according to the procedure described by Dienes et al., 2007, Enzyme and Microbial Technology 40: 1087- 1094.
Variant: The term "variant" means a polypeptide having enzyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
Very high stringency conditions: The term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
Very low stringency conditions: The term "very low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)- linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta- (1 -4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al. , 2005, Adv. Polym. Sci. 186: 1- 67.
In the processes of the present invention, any material containing xylan may be used. In a preferred aspect, the xylan-containing material is lignocellulose.
Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1 ) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyi esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86(1 1 ): 1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601 ; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D- xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321 : 375-381.
Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 (4-(1 , 1 ,3,3- tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μηηοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.
Xylanase: The term "xylanase" means a 1 ,4-beta-D-xylan-xylohydrolase (E.C.
3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylanase activity is determined with 0.2% AZCL- arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μηηοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
Detailed Description of the Invention The present invention relates to enzyme compositions, comprising (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
In one aspect, the enzyme compositions further comprise an Aspergillus fumigatus xylanase, an Aspergillus fumigatus beta-xylosidase, or a combination thereof; or homologs thereof.
The enzyme compositions of the present invention are more efficient in the deconstruction of cellulosic material than a cellulolytic enzyme composition produced by T. reesei.
Enzyme Compositions
In the present invention, any Aspergillus fumigatus cellobiohydrolase I, Aspergillus fumigatus cellobiohydrolase II, an Aspergillus fumigatus beta-glucosidase or variant thereof, Penicillium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity, Aspergillus fumigatus xylanase, or Aspergillus fumigatus beta-xylosidase, or homologs thereof, may be used. In one aspect, the Aspergillus fumigatus cellobiohydrolase I or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and (iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 or the full-length complement thereof.
In another aspect, the Aspergillus fumigatus cellobiohydrolase II or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 or the full-length complement thereof.
In another aspect, the Aspergillus fumigatus beta-glucosidase or a homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6; (iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5; and (iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 or the full-length complement thereof.
In another aspect, the Penicillium sp. (emersonii) GH61 polypeptide having cellulolytic enhancing activity or a homolog thereof is selected from the group consisting of: (i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 8; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and (iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 or the full-length complement thereof.
In another aspect, the Aspergillus fumigatus xylanase or a homolog thereof is selected from the group consisting of: (i) a xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; and (iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; or the full-length complement thereof.
In another aspect, the Aspergillus fumigatus beta-xylosidase or a homolog thereof is selected from the group consisting of: (i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16; (ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 16; (iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 15; and (iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 15 or the full-length complement thereof.
The polynucleotide of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding enzymes according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library may be screened for DNA that hybridizes with the probes described above and encodes an enzyme. Genomic or other DNA may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or 21 ; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, or 15, or the mature polypeptide coding sequence thereof. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; the mature polypeptide thereof; or a fragment thereof.
The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g. , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g. , Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
A protein engineered variant of an enzyme above (or protein) may also be used. In one aspect, the variant is an Aspergillus fumigatus beta-glucosidase variant. In another aspect, the A. fumigatus beta-glucosidase variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512 of SEQ ID NO: 6, wherein the variant has beta-glucosidase activity. In an embodiment, the variant has sequence identity of at least 80%, e.g. , at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, to the amino acid sequence of the parent beta-glucosidase.
In another embodiment, the variant has at least 80%, e.g. , at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 6.
For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 6 is used to determine the corresponding amino acid residue in another beta- glucosidase. The amino acid sequence of another beta-glucosidase is aligned with the mature polypeptide disclosed in SEQ ID NO: 6, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 6 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Identification of the corresponding amino acid residue in another beta-glucosidase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1 -518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1 .83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.
For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g., "Gly205Arg + Ser41 1 Phe" or "G205R + S41 1 F", representing substitutions at positions 205 and 41 1 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively. In one aspect, a variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512. In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 100, 283, 456, and 512. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 100, 283, 456, and 512. In another aspect, a variant comprises a substitution at each position corresponding to positions 100, 283, 456, and 512.
In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 100. In another aspect, the amino acid at a position corresponding to position 100 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In another aspect, the variant comprises or consists of the substitution F100D of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 283. In another aspect, the amino acid at a position corresponding to position 283 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gly In another aspect, the variant comprises or consists of the substitution S283G of the mature polypeptide of SEQ I D NO: 6.
In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 456. In another aspect, the amino acid at a position corresponding to position 456 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu. In another aspect, the variant comprises or consists of the substitution N456E of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 512. In another aspect, the amino acid at a position corresponding to position 512 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Tyr. In another aspect, the variant comprises or consists of the substitution F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 283, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100 and 456, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100 and 512, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 283 and 456, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 283 and 512, such as those described above. In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 456 and 512, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, and 456, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, and 512, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100, 456, and 512, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 283, 456, and 512, such as those described above.
In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 100, 283, 456, and 512, such as those described above.
In another aspect, the variant comprises or consists of one or more (several) substitutions selected from the group consisting of G142S, Q183R, H266Q, and D703G.
In another aspect, the variant comprises or consists of the substitutions F100D +
S283G of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D + N456E of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D + F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions S283G + N456E of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions S283G + F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions N456E +
F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D + S283G + N456E of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D + S283G + F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D + N456E + F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions S283G + N456E + F512Y of the mature polypeptide of SEQ ID NO: 6.
In another aspect, the variant comprises or consists of the substitutions F100D +
S283G + N456E + F512Y of the mature polypeptide of SEQ ID NO: 6. The variants may consist of 720 to 863 amino acids, e.g., 720 to 739, 740 to to 759, 760 to 779, 780 to 799, 800 to 819, 820 to 839, and 840 to 863 amino acids.
The variants may further comprise an alteration at one or more (several) other positions.
The enzyme composition may further comprise one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
One or more (e.g., several) of the enzymes may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) enzymes may be native proteins of a cell, which is used as a host cell to express recombinantly the enzyme composition.
Examples of bacterial endoglucanases that can be used in the present invention, include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No. 5,536,655, WO 00/70031 , WO 05/093050); T ermobifida fusca endoglucanase III (WO 05/093050); and T ermobifida fusca endoglucanase V (WO 05/093050).
Examples of fungal endoglucanases that can be used in the present invention, include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al. , 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GENBANK™ accession no. M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:1 1-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accession no. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555- 563, GENBANK™ accession no. AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, GENBANK™ accession no. Z33381 ), Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439), Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381 ), Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpus albomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurospora crassa endoglucanase (GENBANK™ accession no. XM_324477), Humicola insolens endoglucanase V, Myceliophthora thermophila CBS 1 17.65 endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS 494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6B endoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase, Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestris NRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase, and Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GENBANK™ accession no. M15665).
In one aspect, the enzyme composition further comprises a Trichoderma endoglucanase I. In another aspect, the enzyme composition further comprises a Trichoderma reesei endoglucanase I. In another aspect, the enzyme composition further comprises a Trichoderma reesei Cel7B endoglucanase I (GENBANK™ accession no. M15665). In another aspect, the Trichoderma reesei endoglucanase I is native to the host cell. In another aspect, the the Trichoderma reesei endoglucanase I is the mature polypeptide of SEQ ID NO: 90.
In another aspect, the enzyme composition further comprises a Trichoderma endoglucanase II. In another aspect, the enzyme composition further comprises a Trichoderma reesei endoglucanase II. In another aspect, the enzyme composition further comprises a Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accession no. M19373). In another aspect, the Trichoderma reesei endoglucanase II is native to the host cell. In another aspect, the the Trichoderma reesei endoglucanase I is the mature polypeptide of SEQ ID NO: 92.
The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.
The enzyme composition may also be a fermentation broth formulation or a cell composition.
The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g. , filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
In an embodiment, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
The fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
Host Cells
The present invention also relates to recombinant filamentous fungal host cells, comprising polynucleotides encoding (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (iv) a Penicilllium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
The host cell may be any filamentous fungal cell useful in the recombinant production of an enzyme or protein.
"Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus,
Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et ai, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
In one aspect, the filamentous fungal cell is any Trichoderma cell useful in the recombinant production of an enzyme or protein. For example, the Trichoderma cell may be a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell. In another aspect, the Trichoderma cell is a Trichoderma harzianum cell. In another aspect, the Trichoderma cell is a Trichoderma koningii cell. In another aspect, the Trichoderma cell is a Trichoderma longibrachiatum cell. In another aspect, the Trichoderma cell is a Trichoderma reesei cell. In another aspect, the Trichoderma cell is a Trichoderma viride cell.
In another aspect, the Trichoderma reesei cell is Trichoderma reesei RutC30. In another aspect, the Trichoderma reesei cell is Trichoderma reese/ TV10. In another aspect, the Trichoderma reesei cell is a mutant of Trichoderma reesei RutC30. In another aspect, the Trichoderma reesei cell is mutant of Trichoderma reesei TV10. In another aspect, the Trichoderma reesei cell is a morphological mutant of Trichoderma reesei. See, for example, WO 97/26330, which is incorporated herein by reference in its entirety.
A Trichoderma cell may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al. , 1988, Bio/Technology 6: 1419-1422.
One or more (e.g., several) native cellulase and/or hemicellulase genes may be inactivated in the Trichoderma host cell by disrupting or deleting the genes, or a portion thereof, which results in the mutant cell producing less or none of the cellulase and/or hemicellulase than the parent cell when cultivated under the same conditions. In one aspect, the one or more (e.g., several) cellulase genes encode enzymes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, beta-glucosidase, and swollenin. In another aspect, the one or more (e.g., several) hemicellulase genes encode enzymes selected from the group consisting of xylanase I, xylanase II, xylanase III, and beta-xylosidase. In another aspect, the one or more (e.g., several) hemicellulase genes encode enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, and a mannosidase.
The mutant cell may be constructed by reducing or eliminating expression of a polynucleotide encoding a Trichoderma cellulase or hemicellulase using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. In a preferred aspect, the polynucleotide is inactivated. The polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the polynucleotide. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.
Modification or inactivation of the polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the polynucleotide has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.
Modification or inactivation of the polynucleotide may also be accomplished by insertion, substitution, or deletion of one or more (e.g., several) nucleotides in the gene or a regulatory element required for transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the polynucleotide to be modified, it is preferred that the modification be performed in vitro as exemplified below.
An example of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed. In an aspect, the polynucleotide is disrupted with a selectable marker such as those described herein.
Modification or inactivation of the polynucleotide may also be accomplished by inhibiting expression of an enzyme encoded by the polynucleotide in a cell by administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide encoding the enzyme. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA for inhibiting translation. In another aspect, the double-stranded RNA (dsRNA) molecules comprise a portion of the mature polypeptide coding sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ ID NO: 29 for inhibiting expression of the polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single- stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).
The dsRNAs can be used in gene-silencing to selectively degrade RNA using a dsRNAi of the present invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can be used to generate a loss-of-f unction mutation in a cell, an organ or an animal. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559; 6,51 1 ,824; and 6,515,109. In one aspect, the Trichoderma cellobiohydrolase I or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 18; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 18; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 17; and (iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 17 or the full-length complement thereof.
In another aspect, the Trichoderma cellobiohydrolase II or a homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 20; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 20; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 19; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 19 or the full-length complement thereof.
In another aspect, the Trichoderma beta-glucosidase or a homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 22; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 22; (iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 21 ; and (iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ I D NO: 21 or the full-length complement thereof.
In another aspect, the Trichoderma xylanase or a homolog thereof is selected from the group consisting of: (i) a xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27; and (iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27; or the full-length complement thereof.
In another aspect, the Trichoderma beta-xylosidase or a homolog thereof is selected from the group consisting of: (i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 30; (ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 30; (iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29; and (iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 29 or the full-length complement thereof.
In one aspect, a Trichoderma cellobiohydrolase I gene is inactivated. In another aspect, a Trichoderma cellobiohydrolase II gene is inactivated. In another aspect, a Trichoderma beta-glucosidase gene is inactivated. In another aspect, a Trichoderma xylanase gene is inactivated. In another aspect, a Trichoderma beta-xylosidase gene is inactivated.
In another aspect, a Trichoderma cellobiohydrolase I gene and a Trichoderma cellobiohydrolase II gene are inactivated.
In another aspect, two or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase are inactivated. In another aspect, three or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated. In another aspect, four or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated. In another aspect, five or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta- xylosidase genes are inactivated. In another aspect, six or more (e.g., several) genes selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, beta- glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
In another aspect, the cellobiohydrolase I, cellobiohydrolase II, beta-glucosidase, xylanase I, xylanase II, xylanase III, and beta-xylosidase genes are inactivated.
In another aspect, one or more (e.g., several) protease genes are inactivated. In another aspect, the one or more (e.g., several) protease genes are subtilisin-like serine protease, aspartic protease, and trypsin-like serine protease genes as described in WO 201 1/075677, which is incorporated herein by reference in its entirety. Nucleic Acid Constructs
Nucleic acid constructs comprising a polynucleotide encoding an enzyme or protein can be constructed by operably linking one or more (e.g., several) control sequences to the polynucleotide to direct the expression of the coding sequence in a filamentous fungal host cell under conditions compatible with the control sequences. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a filamentous fungal host cell for expression of a polynucleotide encoding an enzyme or protein. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase {glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,01 1 ,147, which is incorporated herein in its entirety.
The control sequence may also be a transcription terminator, which is recognized by a filamentous fungal host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin- like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase II I, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.
The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by a filamentous fungal host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by a filamentous fungal host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichoderma reesei endoglucanase V.
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into a cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used. Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, and Trichoderma reesei endoglucanase V.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Myceliophthora thermophila laccase (WO 95/33836) and Rhizomucor miehei aspartic proteinase.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of a filamentous fungal host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences include the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.
Expression Vectors
Recombinant expression vectors can be constructed comprising a polynucleotide encoding an enzyme or protein, a promoter, a terminator, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes. Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
The selectable marker may be a dual selectable marker system as described in WO
2010/039889 A2, which is incorporated herein by reference in its entirety. In one aspect, the selectable marker is a hph-tk dual selectable marker system. The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in a filamentous fungal host cell. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of origins of replication useful in a filamentous fungal host cell are AMA1 and ANSI (Gems ef a/., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide may be inserted into a filamentous fungal host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra). Methods of Production
The present invention also relates to methods of producing an enzyme composition, comprising: (a) cultivating a filamentous fungal host cell of the present invention under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
The filamentous fungal host cells are cultivated in a nutrient medium suitable for production of the enzyme composition using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the enzymes to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
The enzymes may be detected using methods known in the art that are specific for the enzyme. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine activity.
The enzymes may be recovered using methods known in the art. For example, the enzyme may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered.
The enzymes may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides. Uses
The present invention is also directed to the following processes for using an enzyme composition of the present invention.
The present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition of the present invention. In one aspect, the processes further comprise recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of the cellulosic material can be separated from insoluble cellulosic material using a method known in the art such as, for example, centrifugation, filtration, or gravity settling.
The present invention also relates to processes of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
The present invention also relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition of the present invention. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.
The processes of the present invention can be used to saccharify the cellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids, alcohols, ketones, gases, and the like). The production of a desired fermentation product from the cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.
The processing of the cellulosic material according to the present invention can be accomplished using methods conventional in the art. Moreover, the processes of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze the cellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of the cellulosic material and the fermentation of sugars to ethanol are combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, i.e. , high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the processes of the present invention.
A conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive stirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor types include fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.
Pretreatment. In practicing the processes of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components of the cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?, Adv. Biochem. Engin. /Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin. /Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. of Mol. Sci. 9: 1621-1651 ; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
The cellulosic material can also be subjected to particle size reduction, sieving, pre- soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.
Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical C02, supercritical H20, ozone, ionic liquid, and gamma irradiation pretreatments.
The cellulosic material can be pretreated before hydrolysis and/or fermentation. Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).
Steam Pretreatment. In steam pretreatment, the cellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g. , hemicellulose, accessible to enzymes. The cellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably performed at 140-250°C, e.g. , 160-200°C or 170-190°C, where the optimal temperature range depends on addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1 -60 minutes, e.g. , 1-30 minutes, 1 -20 minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence time depends on temperature range and addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the cellulosic material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 20020164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.
Chemical Pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments.
A catalyst such as H2S04 or S02 (typically 0.3 to 5% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al. , 2006, Appl. Biochem. Biotechnol. 129- 132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 1 13-1 16: 509-523; Sassner et al. , 2006, Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, the cellulosic material is mixed with dilute acid, typically H2S04, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91 : 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-1 15).
Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).
Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150°C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673- 686). WO 2006/1 10891 , WO 2006/1 10899, WO 2006/1 10900, and WO 2006/1 10901 disclose pretreatment methods using ammonia.
Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151 ; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81 : 1669-1677). The pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.
A modification of the wet oxidation pretreatment method, known as wet explosion
(combination of wet oxidation and steam explosion) can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).
Ammonia fiber explosion (AFEX) involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-150°C and high pressure such as 17- 20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231 ; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121 : 1 133-1 141 ; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). During AFEX pretreatment cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.
Organosolv pretreatment delignifies the cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200°C for 30-60 minutes (Pan et al. , 2005, Biotechnol. Bioeng. 90: 473-481 ; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861 ; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121 : 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.
Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.
In one aspect, the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in the range from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt % acid. The acid is contacted with the cellulosic material and held at a temperature in the range of preferably 140-200°C, e.g., 165-190°C, for periods ranging from 1 to 60 minutes.
In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the cellulosic material is present during pretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
Mechanical Pretreatment or Physical Pretreatment: The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes size reduction of particles. For example, such pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
The cellulosic material can be pretreated both physically (mechanically) and chemically.
Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300°C, e.g., about 140 to about 200°C. In a preferred aspect, mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
Accordingly, in a preferred aspect, the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.
Biological Pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulosic material. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331 ; and Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
Saccharification. In the hydrolysis step, also known as saccharification, the cellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically by an enzyme composition of the present invention.
Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In one aspect, hydrolysis is performed under conditions suitable for the activity of the enzyme(s), i.e., optimal for the enzyme(s). The hydrolysis can be carried out as a fed batch or continuous process where the cellulosic material is fed gradually to, for example, an enzyme containing hydrolysis solution.
The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. For example, the saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 120 hours, e.g., about 16 to about 72 hours or about 24 to about 48 hours. The temperature is in the range of preferably about 25°C to about 70°C, e.g., about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to about 55°C. The pH is in the range of preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is in the range of preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt % or about 20 to about 30 wt %.
In the processes of the present invention, the enzyme composition of the present invention can be added prior to or during fermentation, e.g., during saccharification or during or after propagation of the fermenting microorganism(s).
The enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a Trichoderma host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
The optimum amount of Aspergillus fumigatus cellulases or hemicellulases depends on several factors including, but not limited to, the mixture of component cellulolytic and/or hemicellulolytic enzymes, the cellulosic material, the concentration of cellulosic material, the pretreatment(s) of the cellulosic material, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).
In one aspect, an effective amount of cellulolytic or hemicellulolytic enzyme to the cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1 .25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1 .25 mg, about 0.15 to about 1 .25 mg, or about 0.25 to about 1.0 mg per g of the cellulosic material.
In another aspect, the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, e.g., manganese sulfate.
In another aspect, a GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated cellulosic material such as pretreated corn stover (PCS).
The dioxy compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl moiety as described herein. The dioxy compounds may comprise one or more (e.g., several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives. Non-limiting examples of the dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1 ,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6- dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1 ,2-benzenediol; 4- nitro-1 ,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2- butyne-1 ,4-diol; (croconic acid; 1 ,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1 ,2- propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-butene-1 ,4-diol; 3,4-dihydroxy-3- cyclobutene-1 ,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4- hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate thereof.
The bicyclic compound may include any suitable substituted fused ring system as described herein. The compounds may comprise one or more (e.g., several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally substituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof. Non-limiting examples of the bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.
The heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5-membered heterocycloalkyl or an optionally substituted 5- membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl. Non-limiting examples of the heterocyclic compounds include (1 ,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3-furanone; 5- hydroxy-2(5H)-furanone; [1 ,2-dihydroxyethyl]furan-2,3,4(5H)-trione; ohydroxy-v- butyrolactone; ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconic acid δ- lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)- furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate thereof.
The nitrogen-containing compound may be any suitable compound with one or more (e.g., several) nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety. Non-limiting examples of the nitrogen- containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2- aminophenol; 1 ,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8- tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.
The quinone compound may be any suitable compound comprising a quinone moiety as described herein. Non-limiting examples of the quinone compounds include 1 ,4- benzoquinone; 1 ,4-naphthoquinone; 2-hydroxy-1 ,4-naphthoquinone; 2,3-dimethoxy-5- methyl-1 ,4-benzoquinone or coenzyme Q0; 2,3,5,6-tetramethyl-1 ,4-benzoquinone or duroquinone; 1 ,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1 ,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.
The sulfur-containing compound may be any suitable compound comprising one or more (e.g., several) sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of the sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1 -thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene- 1 ,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.
In one aspect, an effective amount of such a compound described above to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10"6 to about 10, e.g., about 10"6 to about 7.5, about 10"6 to about 5, about 10"6 to about 2.5, about 10"6 to about 1 , about 10"5 to about 1 , about 10"5 to about 10"1 , about 10"4 to about 10"1 , about 10"3 to about 10"1 , or about 10"3 to about 10"2. In another aspect, an effective amount of such a compound described above is about 0.1 μΜ to about 1 M, e.g., about 0.5 μΜ to about 0.75 M, about 0.75 μΜ to about 0.5 M, about 1 μΜ to about 0.25 M, about 1 μΜ to about 0.1 M, about 5 μΜ to about 50 mM, about 10 μΜ to about 25 mM, about 50 μΜ to about 25 mM, about 10 μΜ to about 10 mM, about 5 μΜ to about 5 mM, or about 0.1 mM to about 1 mM. The term "liquor" means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, eic , under conditions as described herein, and the soluble contents thereof. A liquor for cellulolytic enhancement of a GH61 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate by a cellulase preparation. The liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.
In one aspect, an effective amount of the liquor to cellulose is about 10"6 to about 10 g per g of cellulose, e.g., about 10"6 to about 7.5 g, about 10"6 to about 5, about 10"6 to about 2.5 g, about 10"6 to about 1 g, about 10"5 to about 1 g, about 10"5 to about 10"1 g, about 10"4 to about 10"1 g, about 10"3 to about 10"1 g, or about 10"3 to about 10"2 g per g of cellulose.
Fermentation. The fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
In the fermentation step, sugars, released from the cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separate or simultaneous, as described herein.
Any suitable hydrolyzed cellulosic material can be used in the fermentation step in practicing the present invention. The material is generally selected based on the desired fermentation product, i.e. , the substance to be obtained from the fermentation, and the process employed, as is well known in the art.
The term "fermentation medium" is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
"Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e. , convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al, 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeast. Preferred xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting pentose sugars, such as xylose and arabinose, may be genetically modified to do so by methods known in the art.
Examples of bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, 1996, supra).
Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans;
Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;
Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and Zymomonas, such as Zymomonas mobilis.
In a preferred aspect, the yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces spp. In another more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.
In a preferred aspect, the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacilus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.
Commercially available yeast suitable for ethanol production include, e.g., BIOFERM™ AFT and XR (NABC - North American Bioproducts Corporation, GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™ (Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast (Ethanol Technology, Wl, USA).
In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (co- fermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al. , 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61 : 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et al., 1991 , Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).
In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.
It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.
The fermenting microorganism is typically added to the degraded cellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g., about 24 to about 60 hours. The temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
In one aspect, the yeast and/or another microorganism are applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably between about 20°C to about 60°C, e.g. , about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C, and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is preferably applied in amounts of approximately 105 to 1012, preferably from approximately 107 to 1010, especially approximately 2 x 108 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook" (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
A fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A "fermentation stimulator" refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
Fermentation products: A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1 ,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H2), carbon dioxide (C02), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D- gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketide. The fermentation product can also be protein as a high value product.
In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term "alcohol" encompasses a substance that contains one or more (e.g., several) hydroxyl moieties. In a more preferred aspect, the alcohol is n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is 1 ,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer- Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes for fermentative production of xylitol - a sugar substitute, Process Biochemistry 30 (2): 1 17- 124; Ezeji, T. C, Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.
In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or a branched alkane. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is decane. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.
In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, the cycloalkane is cyclopentane. In another more preferred aspect, the cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloheptane. In another more preferred aspect, the cycloalkane is cyclooctane.
In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or a branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.
In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, for example, Richard, A., and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515. In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H2. In another more preferred aspect, the gas is C02. In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V.N. in Biomass and Bioenergy, Vol. 13 (1 -2), pp. 83-1 14, 1997, Anaerobic digestion of biomass for methane production: A review.
In another preferred aspect, the fermentation product is isoprene.
In another preferred aspect, the fermentation product is a ketone. It will be understood that the term "ketone" encompasses a substance that contains one or more (e.g., several) ketone moieties. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diketo-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448.
In another preferred aspect, the fermentation product is polyketide.
Recovery. The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
Examples Strains
Trichoderma reesei strain 981-0-8 (D4) is a mutagenized strain of Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181 : 289- 301 ).
Trichoderma reesei strain AgJgl 15-104-7B1 (PCT/US2010/061 105; WO 201 1/075677) is a T. reesei ku70- derivative of strain 981-0-8 (D4).
Media and Buffer Solutions
2XYT plus ampicillin plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of sodium chloride, 15 g of Bacto agar, and deionized water to 1 liter. One ml of a 100 mg/ml solution of ampicillin was added after the autoclaved medium was cooled to 55°C.
SOC medium was composed of 20 g of Bacto-tryptone, 5 g of Bacto yeast extract, 0.5 g of NaCI, 2.5 ml of 1 M KCI, and deionized water to 1 liter. The pH was adjusted to 7.0 with 10 N NaOH before autoclaving. Then 20 ml of sterile 1 M glucose was added immediately before use.
COVE salt solution was composed of 26 g of KCI, 26 g of MgS04-7H20, 76 g of
KH2P04, 50 ml of COVE trace metals solution, and deionized water to 1 liter.
COVE trace metals solution was composed of 0.04 g of NaB4O7-10H2O, 0.4 g of
CuS04-5H20, 1.2 g of FeS04-7H20, 0.7 g of MnS04-H20, 0.8 g of Na2Mo02-2H20, 10 g of
ZnS04-7H20, and deionized water to 1 liter.
COVE plates were composed of 342.3 g of sucrose, 20 ml of COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCI, 25 g of Noble agar (Difco), and deionized water to
1 liter.
COVE2 plates were composed of 30 g of sucrose, 20 ml of COVE salt solution, 10 ml of 1 M acetamide, 25 g of Noble agar (Difco), and deionized water to 1 liter.
Trichoderma trace metals solution was composed of 216 g of FeCI3-6H20, 58 g of
ZnS04-7H20, 27 g of MnS04-H20, 10 g of CuS04-5H20, 2.4 g of H3B03, 336 g of citric acid, and deionized water to 1 liter. CIM medium was composed of 20 g of cellulose, 10 g of corn steep solids, 1 .45 g of (NH4)2S04, 2.08 g of KH2P04, 0.28 g of CaCI2, 0.42 g of MgS04-7H20, 0.42 ml of Trichoderma trace metals solution, 1-2 drops of antifoam, and deionized water to 1 liter; pH adjusted to 6.0.
YP medium was composed of 10 g of yeast extract, 20 g of Bacto peptone, and deionized water to 1 liter.
PEG buffer was composed of 500 g of polyethylene glycol 4000 (PEG 4000), 10 mM CaCI2, 10 mM Tris-HCI pH 7.5, and deionized water to 1 liter; filter sterilized.
PDA plates were composed of 39 g of Potato Dextrose Agar (Difco) and deionized water to 1 liter.
PDA overlay medium was composed of 39 g of Potato Dextrose Agar (Difco), 2.44 g uridine, and deionized water to 1 liter. The previously autoclaved medium was melted in a microwave and then tempered to 55°C before use.
STC was composed of 1 M sorbitol, 10 mM mM CaCI2, and 10 mM Tris-HCI, pH 7.5; filter sterilized.
TE buffer was composed of 1 M Tris pH 8.0 and 0.5 M EDTA pH 8.0.
20X SSC was composed of 175.3 g of NaCI, 88.2 g of sodium citrate, and deionized water to 1 liter.
TrMM-G medium was composed of 20 ml of COVE salt solution, 6 g of (NH4)2S0 , 0.6 g of CaCI2, 25 g of Nobel agar (Difco), 20 g of glucose, and deionized water to 1 liter.
NZY+ medium was composed of 5 g of NaCI, 3 g of MgS04-7H20, 5 g of yeast extract, 10 g of NZ amine, 1.2 g of MgCI2, 4 g of glucose, and deionized water to 1 liter.
Example 1 : Construction of a Trichoderma reesei cbhl-Aspergillus fumigatus cbhl replacement construct pJfySI 39
The Aspergillus fumigatus cellobiohydrolase I (cbhl) coding sequence (SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid sequence]) was amplified from pEJG93 (WO 201 1/057140) using the gene-specific forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion for an I N- FUSION® reaction and the underlined portion is an introduced Pac I site.
Forward primer:
5'-cgcggacigcgcaccATGCTGGCCTCCACCTTCTCCTACC-3' (SEQ ID NO: 31 )
Reverse primer:
5'-ctttcgccacggagcJtaaJtaaC7/\C/\GGC/\C7G/\G/\G7/\/\7/\/\7C/\-3' (SEQ ID NO: 32)
The amplification reaction was composed of 20 ng of pEJG93, 200 μΜ dNTP's, 0.4 μΜ primers, 1X HERCULASE® Reaction Buffer (Stratagene, La Jolla, CA, USA), and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA, USA) in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S (Eppendorf Scientific, Inc., Westbury, NY, USA) programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 7 minutes. The PCR products were separated by 1 % agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) buffer where a 1.6 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol.
The 1.6 kb PCR product was inserted into Nco \IPac l-digested pSMai155 (WO 05/074647) using an IN-FUSION® Advantage PCR Cloning Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 1X I N-FUSION® Reaction Buffer (Clontech, Palo Alto, CA, USA), 125 ng of Nco \IPac I- digested pSMai155, 100 ng of the 1.6 kb PCR product, and 1 μΙ of IN-FUSION® Enzyme (Clontech, Palo Alto, CA, USA) in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C followed by 15 minutes at 50°C. After the incubation period 40 μΙ of TE buffer were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by sequencing and one clone containing the insert with no PCR errors was identified and designated pJfyS139-A. Plasmid pJfyS139-A was used for insertion of the Herpes simplex virus thymidine kinase (tk) gene.
The Herpes simplex virus tk coding sequence (SEQ ID NO: 33 [DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) was liberated from pJfySI 579-8-6 (WO 2010/039840) by digesting the plasmid with Bgl II and Bam HI. The digestion was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 2.3 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The tk gene cassette was inserted into Bam Hl-digested, calf intestine phosphatase-treated pJfyS139-A using a QUICK LIGATION™ Kit (New England Biolabs, Inc., Ipswich, MA USA) according to the manufacturer's protocol. The ligation reaction was composed of 50 ng of the Bam Hl- digested, calf intestine phosphatase-treated pJfyS139-A, 50 ng of the 2.3 kb tk gene insert, 1 X QUICK LIGATION™ Buffer (New England Biolabs, Inc., Ipswich, MA USA), and 5 units of QUICK LIGASE™ (New England Biolabs, Inc., Ipswich, MA USA) in a final volume of 20 μΙ. The reaction was incubated at room temperature for 5 minutes and 2 μΙ of the reaction were used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Xma I to determine the presence and orientation of the insert and a clone containing the insert was identified and designated pJfyS139-B. Plasmid pJfyS139-B was used for insertion of a T. reesei 3' cbhl gene flanking sequence.
The 3' cbhl gene flanking sequence was amplified from T. reesei RutC30 genomic DNA using the forward and reverse primers below. The underlined portion represents an introduced Not I site for cloning.
Forward Primer:
5'-ttaqactqcqqccqcGTGGCGAAAGCCTGACGCACCGGTAGAT-3' (SEQ ID NO: 35)
Reverse Primer:
5'-aqtaqttaqcqqccqcACGGCACGGTTAAGCAGGGTCTTGC-3' (SEQ ID NO: 36)
Trichoderma reesei RutC30 was grown in 50 ml of YP medium supplemented with 2% glucose (w/v) in a 250 ml baffled shake flask at 28°C for 2 days with agitation at 200 rpm. Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, La Jolla, CA, USA), washed twice in deionized water, and frozen under liquid nitrogen. Frozen mycelia were ground by mortar and pestle to a fine powder. Total DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia, CA, USA) with the lytic incubation extended to 2 hours.
The amplification reaction was composed of 150 ng of T. reesei RutC30 genomic DNA, 200 μΜ dNTP's, 0.4 μΜ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes.
The PCR reaction was subjected to a MINELUTE® Nucleotide Removal Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol. The resulting PCR mixture was digested with Not I and the digested PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer. A 1.3 kb fragment containing the 3' cbhl gene flanking sequence was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The 1 .3 kb fragment was inserted into Not l-linearized, calf intestine phosphatase-treated pJfyS139-B using a QUICK LIGATION™ Kit. The QUICK LIGATION™ reaction was composed of 100 ng of the Not l-linearized, calf intestine phosphatase-treated pJfyS139-B, 20 ng of the 1.3 kb fragment, 1 X QUICK LIGATION™ Buffer, and 5 units of QUICK LIGASE™ in a final volume of 20 μΙ. The reaction was incubated at room temperature for 5 minutes and 2 μΙ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Xma I to determine the presence and orientation of the insert and positive clones were sequenced. A clone containing the 3' cbhl gene flanking sequence with no PCR errors was designated pJfyS139 (Figure 1 ). Plasmid pJfyS139 was used as the vector to replace the T. reesei cbhl gene.
Example 2: Trichoderma reesei protoplast generation and transformation
Protoplast preparation and transformation were performed using a modified protocol by Penttila et al. , 1987, Gene 61 : 155-164. Briefly, Trichoderma reesei strain AgJgl 15-104- 7B1 (PCT/US2010/061 105, WO 201 1/075677) was cultivated in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine at 27°C for 17 hours with gentle agitation at 90 rpm. Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System (Millipore, Bedford, MA, USA) and washed twice with deionized water and twice with 1.2 M sorbitol. Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX® 200 G (Novozymes A/S, Bagsvaerd, Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St. Louis, MO, USA) per ml for 15-25 minutes at 34°C with gentle shaking at 90 rpm. Protoplasts were collected by centrifuging for 7 minutes at 400 x g and washed twice with cold 1 .2 M sorbitol. The protoplasts were counted using a haemocytometer and re-suspended to a final concentration of 1x108 protoplasts per ml in STC. Excess protoplasts were stored in a Cryo 1 °C Freezing Container (Nalgene, Rochester, NY, USA) at -80°C.
Approximately 100 μg of a transforming plasmid described in the following Examples were digested with Pme I. The digestion reaction was purified by 1 % agarose gel electrophoresis using TAE buffer. A DNA band was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA). The resulting purified DNA was added to 100 μΙ of the protoplast solution and mixed gently. PEG buffer (250 μΙ) was added, mixed, and incubated at 34°C for 30 minutes. STC (3 ml) was then added, mixed, and spread onto PDA plates supplemented with 1 M sucrose. After incubation at 28°C for 16 hours, 20 ml of an overlay PDA medium supplemented with 35 μg of hygromycin B per ml were added to each plate. The plates were incubated at 28°C for 4-7 days. Example 3: Replacement of the native Trichoderma reesei cbhl gene with the Aspergillus fumigatus cbhl coding sequence
In order to replace the Trichoderma reesei native cbhl gene (SEQ ID NO: 17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid sequence]) with the Aspergillus fumigatus cbhl coding sequence (SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid sequence]), Trichoderma reesei ku70- strain AgJgl 15-104-7B1 (PCT/US2010/061 105, WO 201 1/075677) was transformed with 4 x 2 μg of Pme l-linearized pJfyS139 (Example 1 ) according to the procedure described in Example 2. Seven transformants were obtained and each one was picked and transferred to a PDA plate and incubated for 7 days at 28°C. Genomic DNA was isolated from the transformants according to the procedure described in Example 1 and each transformant submiited to Southern analysis.
For Southern analysis, 2 μg of genomic DNA was digested with 33 units of Bgl II in a 50 μΙ reaction volume and subjected to 1% agarose electrophoresis in TAE buffer. The DNA in the gel was depurinated with one 10 minute wash in 0.25 N HCI, denatured with two 15 minute washes in 0.5 N NaOH-1.5 M NaCI, neutralized with one 30 minute wash in 1 M Tris pH 8-1.5 M NaCI, and incubated in 20X SSC for 5 minutes. The DNA was transferred to a NYTRAN® Supercharge membrane (Whatman, Inc., Florham Park, NJ, USA) using a TURBOBLOTTER™ System (Whatman, Inc., Florham Park, NJ, USA) according to the manufacturer's protocol. The DNA was UV crosslinked to the membrane using a STRATALINKER™ UV Crosslinker (Stratagene, La Jolla, CA, USA) and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb (Roche Diagnostics Corporation, Indianapolis, IN, USA).
A probe hybridizing to the 3' cbhl gene flanking sequence was generated using a PCR Dig Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions with the forward and reverse primers shown below. The PCR reaction was composed of 1X HERCULASE® Reaction Buffer, 400 nM of each primer, 200 μΜ DIG-labeled dUTP-containing dNTPs, 20 ng of pJfyS139, and 1.5 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds; and 1 cycle at 72°C for 7 minutes.
Forward primer:
5'-AAAAAACAAACATCCCGTTCATAAC-3' (SEQ ID NO: 37)
Reverse primer:
5'- AAC AAG GTTT AC C G GTTT C G AAAAG-3 ' (SEQ ID NO: 38) The probe was purified by 1 % agarose gel electrophoresis using TAE buffer where a 0.5 kb band corresponding to the probe was excised from the gel and extracted using a MI NELUTE® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42°C for 15-17 hours. The membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C. The probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that 3 of the 7 transformants contained the replacement cassette at the cbhl locus and one transformant, T. reesei JfyS 139-8, was chosen for curing the hpt and tk markers.
A fresh plate of spores was generated by transferring spores of a 7 day old PDA plate grown at 28°C to a PDA plate and incubating for 7 days at 28°C. Spores were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemocytometer and 105 spores were spread onto 150 mm plates containing TrMM-G medium supplemented with 1 μΜ 5-fluoro-2'-deoxyuridine (FdU).
Three hundred FdU-resistant spore isolates were obtained and DNA was extracted from 2 of the spore isolates as described above. The isolates were submiited to Southern analysis as described above and the results indicated that both spore isolates had excised the hpt/tk region between the homologous repeats of the replacement cassette. One strain designated T. reesei JfyS139-8A was chosen for replacing the cbhll gene.
Example 4: Construction of an empty Trichoderma reesei cbhll replacement construct pJfyS142
To generate a construct to replace the Trichoderma reesei cbhll gene (SEQ ID NO: 19 [DNA sequence] and SEQ ID NO: 20 [deduced amino acid sequence]) with the Aspergillus fumigatus cbhll coding sequence (SEQ ID NO: 3 [DNA sequence] and SEQ ID NO: 4 [deduced amino acid sequence]), the T reesei cbhll promoter was first amplified from T reesei RutC30 genomic DNA using the gene-specific forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion in an INFUSION® reaction. T reesei RutC30 genomic DNA was prepared according to the procedure described in Example 1.
Forward primer:
5'-acgaaiigiiiaaacgicgacCCAAGTATCCAGAGGTGTATGGAAATATCAGAT-3' (SEQ ID NO: 39)
Reverse primer: 5'-cgcgiagaicigcggccaiGGTGCAATACACAGAGGGTGATCTT-3' (SEQ ID NO: 40)
The amplification reaction was composed of 20 ng of T. reesei RutC30 genomic DNA, 200 μΜ dNTP's, 0.4 μΜ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes. The PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 1 .6 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
The 1.6 kb PCR product was inserted into Nco \/Sal l-digested pSMai155 (WO
05/074647) using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 125 ng of the Nco \ISal l-digested pSMai155, 100 ng of the 1.6 kb PCR product, and 1 μΙ of IN-FUSION® Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Pci I and positive clones sequenced to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pJfyS142-A. Plasmid pJfyS142-A was used to insert the T. reesei cbhll terminator.
The cbhll terminator was amplified from T. reesei RutC30 genomic DNA using the gene-specific forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion in an IN-FUSION® reaction.
Forward primer:
5'-aiciacgcgiaciagiiaaiiaaGGCTTTCGTGACCGGGCTTCAAACA-3' (SEQ ID NO: 41 ) Reverse primer:
5'-gcggccgiiaciagiggaiccACTCGGAGTTGTTATACGCTACTCG-3' (SEQ ID NO: 42)
The amplification reaction was composed of 150 ng of T. reesei RutC30 genomic DNA, 200 μΜ dNTP's, 0.4 μΜ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 25 cycles each at 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 50 seconds; and 1 cycle at 72°C for 7 minutes. PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 0.3 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
The 0.3 kb PCR product was inserted into Pac \IBam Hl-digested pJfyS142-A using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 150 ng of the Pacl/Bam Hl-digested pJfyS142-A, 50 ng of the 0.3 kb PCR product, and 1 μΙ of INFUSION® Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The transformants were screened by sequence analysis to identify positive clones and to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pJfyS142-B. Plasmid pJfyS142-B was used for insertion of the Herpes simplex tk gene.
The Herpes simplex tk gene was liberated from pJfySI 579-8-6 (WO 2010/039840) by digesting the plasmid with Bgl II and Bam HI. The digestion was submitted to 1 % agarose gel electrophoresis using TAE buffer where a 2.3 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The tk cassette was inserted into Bam Hl- digested, calf Intestine phoshatase-dephosphorylated pJfyS142-B using a QUICK LIGATION™ Kit according to the manufacturer's protocol. The ligation reaction was composed of 50 ng of the Bam Hl-digested, calf Intestine phoshatase-dephosphorylated pJfyS142-B, 50 ng of the 2.3 kb ί/ gene insert, 1 X QUICK LIGATION™ Buffer, and 5 units of QUICK LIGASE™ in a 20 μΙ ligation volume. The reaction was incubated at room temperature for 5 minutes and 2 μΙ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Xma I and Bam HI to determine the presence and orientation of the insert and a clone containing the insert was identified and designated pJfyS142-C. Plasmid pJfyS142-C was used for insertion of the T. reesei 3' cbhll gene flanking sequence.
The 3' cbhll gene flanking sequence was amplified from T. reesei RutC30 genomic
DNA using the forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion in an IN-FUSION® reaction. Forward primer:
5'-aiccaicacaciggcggccgcGCTTCAAACAATGATGTGCGATGGT-3' (SEQ I D NO: 43) Reverse primer:
5'-ga tgca tgctcgagcggccgcCJ AC CTTG G CAG C C CT AC G AG AG AG-3 ' (SEQ ID NO: 44)
The amplification reaction was composed of 150 ng of T. reesei RutC30 genomic
DNA, 200 μΜ dNTP's, 0.4 μΜ primers, 1X HERCULASE® Reaction Buffer, and 1 .875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute 50 seconds; and 1 cycle at 72°C for 7 minutes. The PCR reaction was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 1 .5 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The 3' cbhll gene flanking sequence was inserted into Not l-linearized pJfyS142-C using an INFUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN- FUSION® reaction was composed of 1 X IN-FUSION® Reaction Buffer, 150 ng of pJfyS142- C, 80 ng of the 1.5 kb PCR product, and 1 μΙ of IN-FUSION® Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Bgl II and positive clones were sequenced to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pJfyS142 (Figure 2). Plasmid pJfyS142 was used to insert the A. fumigatus cbhll coding sequence.
Example 5: Construction of a Trichoderma reesei cbhll-Aspergillus fumigatus cbhll replacement construct pJfyS144
The Aspergillus fumigatus cbhll coding sequence (SEQ ID NO: 3 [DNA sequence] and SEQ ID NO: 4 [deduced amino acid sequence]) was amplified from pAILo33 (WO 201 1/057140) using the forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion for an IN-FUSION® reaction.
Forward primer:
5'-cicigigiaiigcaccATGAAGCACCTTGCATCTTCCATCG-3' (SEQ ID NO: 45)
Reverse primer:
5'-ccggicacgaaagccTT AATTAAAAGGACGGGTTAGCGTT-3' (SEQ ID NO: 46) The amplification reaction was composed of 20 ng of pAILo33, 200 μΜ dNTP's, 0.4 μΜ primers, 1 mM HERCULASE® Reaction Buffer, and 1.875 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes; and 1 cycle at 72°C for 7 minutes.
The PCR reaction was subjected to 1 % agarose gel electrophoresis using TAE buffer where a 1.7 kb band was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The 1 .7 kb PCR product was inserted into Nco \IPac l-digested pJfyS142 (Example 4) using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 120 ng of the Nco \IPac l-digested pJfyS142, 70 ng of the 1.7 kb PCR product, and 1 μΙ of IN-FUSION® Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were sequenced to ensure the absence of PCR errors and determine the presence of the insert. One clone with error-free sequence was identified and designated pJfyS144 (Figure 3). Plasmid pJfyS144 was used to replace the native cbhll gene with the cbhll coding sequence from A. fumigatus.
Example 6: Replacement of the native Trichoderma reesei cbhll gene with the Aspergillus fumigatus cbhll coding sequence
In order to replace the native T. reesei cbhll gene (SEQ ID NO: 19 [DNA sequence] and SEQ ID NO: 20 [deduced amino acid sequence]) with the Aspergillus fumigatus cbhll coding sequence (SEQ ID NO: 3 [DNA sequence] and SEQ ID NO: 4 [deduced amino acid sequence]), Trichoderma reesei JfyS139-8A (Example 3) was transformed according to the procedure described in Example 2 with 2 μg of Pme l-linearized and gel purified pJfyS144 (Example 5). Seven transformants were obtained and each one was picked and transferred to a PDA plate and incubated for 7 days at 28°C. A fungal spore PCR method described below was used to screen for transformants bearing gene replacement using the forward primer shown below annealing to a region upstream of the 5' cbhll gene flanking sequence beyond the region of integration, and the reverse primer shown below annealing in the A. fumigatus cbhll coding sequence.
Forward primer: 5'-AGCCACATGCCGCATATTGACAAAG-3' (SEQ ID NO: 47)
Reverse primer:
5'-AGGGATTCAGTGTGCTACAGGCTGC-3' (SEQ ID NO: 48)
A 1.8 kb PCR product would be generated only upon the occurrence of a precise gene replacement at the cbhll locus. If the cassette had integrated elsewhere in the genome, no amplification would result.
A small amount of spores from each transformant was suspended in 25 μΙ of TE buffer and heated on high in a microwave oven for 1 minute. Each microwaved spore suspension was used as a template in the PCR reaction. The reaction was composed of 1 μΙ of the microwaved spore suspension, 1 μΙ of a 10 mM dNTPs, 12.5 μΙ of 2X ADVANTAGE® GC-Melt LA Buffer (Clontech, Mountain View, CA, USA), 25 pmol of forward primer, 25 pmol of reverse primer, 1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix (Clontech, Mountain View, CA, USA), and 9.25 μΙ of water. The reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 35 cycles each at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute 40 seconds; 1 cycle at 72°C for 7 minutes; and a 4°C hold. The PCR reactions were subjected to 1 % agarose gel electrophoresis using TAE buffer. The spore PCR indicated that four of the seven transformants contained the replacement cassette at the targeted locus and three of them were submitted to Southern analysis to confirm the replacement cassette was in a single copy.
Genomic DNA was isolated from the three transformants according to the procedure described in Example 1 and each transformant submitted to Southern analysis. For Southern analysis, 2 μg of genomic DNA was digested with 50 units of Dra I in a 50 μΙ reaction volume and subjected to 1 % agarose electrophoresis in TAE buffer. The DNA in the gel was depurinated with one 10 minute wash in 0.25 N HCI, denatured with two 15 minute washes in 0.5 N NaOH-1 .5 M NaCI, neutralized with one 30 minute wash in 1 M Tris pH 8-1 .5 M NaCI, and incubated in 20X SSC for 5 minutes. The DNA was transferred to a NYTRAN® Supercharge membrane. The DNA was UV crosslinked to the membrane using a STRATALINKER™ UV crosslinker and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb.
A probe hybridizing to the 3' cbhll gene flanking sequence was generated using a PCR Dig Probe Synthesis Kit according to the manufacturer's instructions with the forward and reverse primers indicated below. The PCR reaction was composed of 1X HERCULASE® Reaction Buffer, 400 nM each primer, 200 μΜ DIG-labeled dUTP-containing dNTPs, 150 ng of T. reesei RutC30 genomic DNA, and 1 .5 units of HERCULASE® Hot Start High-Fidelity DNA Polymerase. The reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 51 °C for 30 seconds, and 72°C for 40 seconds; and 1 cycle at 72°C for 7 minutes.
Forward primer:
5'-AAAAAACAAACATCCCGTTCATAAC-3' (SEQ ID NO: 49)
Reverse primer:
5'-AACAAGGTTTACCGGTTTCGAAAAG-3' (SEQ ID NO: 50)
The probe was purified by 1 % agarose gel electrophoresis using TAE buffer where a 0.5 kb band corresponding to the probe was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42°C for approximately 17 hours. The membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C. The probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that the three transformants contained the replacement cassette at the cbhll locus and all three (designated JfyS 139/144-5, -6, and -10) were chosen for curing the hpt and tk markers.
A fresh plate of spores for each transformant was generated by transferring a plug of a 7 day old culture grown on a PDA plate at 28°C to a new PDA plate and incubating for 7 days at 28°C. Spores were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemacytometer and 105 and 104 spores were spread onto 150 mm plates containing TrMM-G medium supplemented with 1 μΜ FdU.
Approximately 500 FdU-resistant spore isolates for each transformant were obtained from the plate containing 105 spores and approximately 100 FdU-resistant spore isolates for each transformant from the plate containing 104 spores. Eight spore isolates were picked for strains JfyS139/144-5 and -6 and four were picked for strain JfyS139/144-10. Each isolate 1 to 8 from primary transformant 5 was designated JfyS 139/144-5A to -5H. Isolates 1 to 8 from primary transformant 6 were designated JfyS 139/144-6A to 6H. Isolates from primary transformant 10 were designated JfyS139/144-1 OA to 10D for isolates 1 to 4. Spore PCR was conducted as described above, using the forward and reverse primers shown below, to confirm the hpt and tk markers had been correctly excised.
Forward primer:
5'-GTTAAGCATACAATTGAACGAGAATGG-3' (SEQ ID NO: 51 )
Reverse primer:
5'-GATGATATAATGGAGCAAATAAGGG-3' (SEQ ID NO: 52) The PCR reactions were performed as described above with the following cycling parameters: 1 cycle at 95°C for 2 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 6 minutes seconds; and 1 cycle at 72°C for 7 minutes.
The primers annealed to the 5' (forward) and 3' (reverse) flanking sequences used for the cbhll gene replacement. Strains from which the hpt/tk cassette had been correctly excised would display a 3.5 kb fragment while those with the markers intact would display an 8 kb fragment. The PCR screen indicated that all of the spore isolates had correctly excised the hpt/tk cassette.
DNA was extracted from the A and B spore isolates from each primary transformant and submiited to Southern analysis as described above. The Southern analysis confirmed that each spore isolate had correctly excised the hpt/tk cassette. Spore isolate T. reesei JfyS139/144-10B was chosen to represent the strain containing both the T. reesei cbhl and cbhll genes replaced with the respective homologs from Aspergillus fumigatus. Example 7: Generation of Trichoderma reesei ku70 gene repair plasmid pTH239
Four DNA segments were combined using an IN-FUSION® Advantage PCR Cloning Kit to generate a construct to replace the disrupted Trichoderma reesei ku70 coding sequence with the native Trichoderma reesei ku70 coding sequence [(SEQ ID NO: 53 [DNA sequence] and SEQ ID NO: 54 [deduced amino acid sequence]). The ampicillin resistance marker region including the prokaryotic origin of replication was amplified from pJfyS139-B (Example 4) using the sequence-specific forward and reverse primers shown below (SEQ ID NOs: 55 and 56). The T reesei ku70 gene upstream sequence (consisting of 989 bp from upstream of the ku 70 coding sequence and the first 1010 bp of the ku 70 coding sequence) was amplified from T reesei 981 -0-8 genomic DNA using the sequence-specific forward and reverse primers shown below (SEQ ID NOs: 57 and 58). The T reesei ku70 gene downstream sequence (consisting of a 500 bp segment repeated from the 3' end of the 1010 bp segment of the ku70 coding sequence amplified in the upstream PCR product, and a 1067 bp segment containing the remainder of the ku 70 coding sequence, and 461 bp from downstream of the ku 70 coding sequence) was amplified from T reesei 981 -0-8 genomic DNA using the sequence-specific forward and reverse primers shown below (SEQ ID NOs: 59 and 60). T reesei 981-0-8 genomic DNA was prepared according to the procedure described in Example 1.
Forward primer:
5'-GTGTGCGGCCGCTCGAGCATGCATGTTTAAACAGCTTGGCACTGGCCGTCGTTTT-3' (SEQ ID NO: 55)
Reverse primer: 5'-ATCAGCCCCGAGACGGCGCCGCGTTTAAACAATTCGTAATCATGGTCATAGCTGT-3' (SEQ ID NO: 56)
Forward primer:
5'-CATGATTACGAATTGTTTAAACGCGGCGCCGTCTCGGGGCTGATCTTGTCGAGGA-3' (SEQ ID NO: 57)
Reverse primer:
5'-GGCGGCCGTTACTAGTGGATCCAGCCCTTGACAGTGATCTTGAGTCCAGGTGCAA-3' (SEQ ID NO: 58)
Forward primer:
5'-TGCAGATATCCATCACACTGGCGGCCGCAGTTTCCATGTCCAACGTGTTGTTTTGCG C-3' (SEQ ID NO: 59)
Reverse primer:
5'-GCCAGTGCCAAGCTGTTTAAACATGCATGCTCGAGCGGCCGCACACGCCCTCTCCT CG-3' (SEQ ID NO: 60)
For amplification of the ampicillin resistance marker and prokaryotic origin of replication region, the reaction was composed of 100 ng of T. reesei 981-0-8 genomic DNA, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NO: 55 and 56), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer (New England Biolabs, Inc., Ipswich, MA, USA), and 1 .0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA) in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes. The PCR product was separated by 1 % agarose gel electrophoresis using TAE buffer where a 2.692 kb fragment was excised from the gels and extracted using a MINELUTE® Gel Extraction Kit.
For amplification of the ku 70 gene upstream sequence or downstream sequence, the reactions were composed of 100 ng of pJfyS139-B, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NOs: 57 and 58 or 59 and 60, respectively), 1 X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds; and 1 cycle at 72°C for 7 minutes. The PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where 1 .999 kb and 2.028 kb fragments were separately excised from the gels and extracted using a MINELUTE® Gel Extraction Kit.
The fourth DNA segment was generated from a restriction enzyme digestion of pJfyS139-B with Not I and Bam HI. The reaction was composed of 5 μg of pJfyS139-B, 10 units of Not I, 20 units of Bam HI, and 20 μΙ of Restriction Enzyme Buffer 2 (New England Biolabs, Inc., Ipswich, MA, USA) in a total volume of 50 μΙ. The reaction was incubated for 1 hour at 37°C and then separated by 1 % agarose gel electrophoresis using TAE buffer where a 4.400 kb fragment was excised from the gel and extracted using a MINELUTE® Gel Extraction Kit.
The three PCR products of 2,028 bp, 1 ,999 bp and 2,692 bp were inserted into Not I and Bam Hl-digested pJfyS139-B using an IN-FUSION® Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 1X IN-FUSION® Reaction Buffer, 50 ng of the Not \l Bam Hl-digested pJfyS139-B, 50 ng of the 1.999 kb ku 70 gene upstream PCR product, 50 ng of the 2.028 kb ku 70 gene downstream PCR product, 50 ng of the 2.692 kb ampicillin resistance marker and prokaryotic origin of replication PCR product, and 1 μΙ of IN-FUSION® Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C followed by 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 3 μΙ aliquot was used to transform E. coli XL10 GOLD® competent cells (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and then 500 μΙ of NZY+ medium, pre-heated to 42°C, were added. The tubes were incubated at 37°C with shaking at 200 rpm for 40 minutes and then plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting transformants were screened by restriction digestion analysis with Hind III and Xba I and positive clones sequenced to ensure the absence of PCR errors. One clone containing the insert with no PCR errors was identified and designated pTH239.
Example 8: Repair of the ku70 gene in the A. fumigatus cbhl and cbh2 replacement strain JfyS139/144-10B
The native Trichoderma reesei ku70 gene was repaired in strain T. reesei JfyS139/144-10B (Example 6) in order to facilitate strain manipulation steps requiring the function of the ku 70 gene in non-homologous end-joining. T. reesei JfyS129/144-10B was transformed with 23 x 2 of Pme l-linearized pTH239 (Example 7) according to the procedure described in Example 2. Nineteen transformants were obtained and each one was separately transferred to a PDA plate and incubated for 7 days at 28°C.
All nineteen transformants were screened by PCR to confirm homologous integration of the pTH239 Pme I fragment at the disrupted ku70 gene locus. For each of the transformants a sterile inoculation loop was used to collect spores from a 7 day old PDA plate. The spores were transferred to a tube containing 25 μΙ of 1 mM EDTA-10 mM Tris buffer and microwaved on high for 1 minute. A 1 μΙ aliquot of the microwaved spore mixture was added directly to the PCR reaction as template DNA. A set of PCR primers shown below were designed to amplify across the disrupted region of the ku70 coding sequence to distinguish between the host genome with the disruption in the ku70 coding sequence (848 bp) and the pTH239 targeted strain of interest (606 bp). The PCR reaction was composed of 1X ADVANTAGE® Genomic LA Polymerase Reaction Buffer (Clontech, Mountain View, CA, USA), 400 nM of each primer, 200 μΜ dNTPs, 1 μΙ of microwaved TE-spore mixture (described above), and 1.0 unit of ADVANTAGE® Genomic LA Polymerase (Clontech, Mountain View, CA, USA). The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 30 cycles each at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds; and 1 cycle at 72°C for 7 minutes.
Forward primer:
5'-CAATGACGATCCGCACGCGT-3' (SEQ ID NO: 61 )
Reverse primer:
5'- CAATGACGATCCGCACGCGT-3' (SEQ ID NO: 62)
Only one of the nineteen transformants (#19) was positive for the 606 bp PCR product and negative for the 848 bp PCR product indicative of a strain containing the pTH239 Pme\ fragment homologously integrated at the ku 70 locus.
Spores from the 7 day old PDA plate of transformant #19 were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemocytometer and 106 spores were spread onto 150 mm plates containing TrMM-G medium supplemented with 1 μΜ 5-fluoro-2'-deoxyuridine (FdU) and cultured for 5 days at 28°C. Twenty-two FdU-resistant spore isolates were obtained and transferred to PDA plates and cultivated at 28°C for five days.
All twenty-two spore isolates (#19A-V) were screened by PCR for excision of the hpt/tk marker region present between the homologous repeats of the ku 70 coding sequence within the repair cassette. For each of the spore isolates a sterile inoculating loop was used to collect spores from a 7 day old PDA plate. The spores were transferred to a tube containing 25 μΙ of 1 mM EDTA-10 mM Tris buffer and microwaved on high for 1 minute. A 1 μΙ aliquot of the spore mixture was added directly to the PCR reaction as template genomic DNA. A set of PCR primers shown below were designed to amplify across the hpt/tk region to distinguish between the presence (6 kb) or absence (1 .1 kb) of the hpt/tk region. The PCR reaction was composed of 1X ADVANTAGE® Genomic LA Polymerase Reaction Buffer, 400 nM of each primer (below), 200 μΜ dNTPs, 1 μΙ of microwaved TE-spore mixture (described above), and 1.0 unit of ADVANTAGE® Genomic LA Polymerase. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 95°C for 10 minutes; 30 cycles each at 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 6 minutes; and 1 cycle at 72°C for 7 minutes. Forward primer:
5'-GACACTCTTTTCTCCCATCT-3' (SEQ ID NO: 63)
Reverse primer:
5'-GAGGAGCAGAAGAAGCTCCG-3' (SEQ ID NO: 64)
All twenty-two spore isolates were negative for the 6 kb PCR product corresponding to the hpt/tk marker region.
Spores from the 7 day old PDA plates of isolates #19A and #19L were collected in 10 ml of 0.01 % TWEEN® 20 using a sterile spreader. The concentration of spores was determined using a hemocytometer and 103, 102, and 101 spores were spread onto 150 mm PDA plates containing 1 M sucrose and cultured for 3 days at 28°C. Ten spore isolates were selected from the PDA plates for both strains #19A and #19L and transferred to fresh PDA plates and placed at 28°C.
Genomic DNA was extracted from 6 spore isolates of both #19L and #19A, according to the procedure described in Example 1 and submiited to Southern analysis.
For Southern analysis, 2 μg of genomic DNA was digested with (1 ) 5 units and 10 units, respectively, of Asc I and Xho I or (2) 5 units and 25 units, respectively, of Asc I and Apa I in a 50 μΙ reaction volume and subjected to 1 % agarose electrophoresis using TAE buffer. The DNA in the gel was depurinated with one 10 minute wash in 0.25 N HCI, denatured with two 15 minute washes in 0.5 N NaOH-1.5 M NaCI, neutralized with one 30 minute wash in 1 M Tris pH 8-1 .5 M NaCI, and incubated in 20X SSC for 5 minutes. The DNA was transferred to a NYTRAN® Supercharge membrane using a TURBOBLOTTER™ System according to the manufacturer's protocol. The DNA was UV crosslinked to the membrane using a STRATALINKER™ UV Crosslinker and prehybridized for 1 hour at 42°C in 20 ml of DIG Easy Hyb.
A probe hybridizing to the 3' end of the ku 70 coding sequence was generated using a
PCR Dig Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions with the forward and reverse primers shown below. In order to generate a pure template for the probe PCR reaction, the 3' end of the ku70 coding sequence was amplified from T. reesei 981-0-8 genomic DNA. The PCR reaction was composed of 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, 1 μΜ of each primer, 200 μΜ dNTPs, 165 ng of T. reesei 981 -0-8 genomic DNA, and 1 .0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 35 cycles each at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 15 seconds; and 1 cycle at 72°C for 10 minutes.
Forward primer:
5'-gcatatataacccactcaagta-3' (SEQ ID NO: 65) Reverse primer:
5'-attatcttggaccggccgcagg-3' (SEQ ID NO: 66)
The 0.5 kb probe template was purified by 1 % agarose gel electrophoresis using TAE buffer and excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The purified PCR product was used to generate a DIG-labeled probe as specified by the manufacturer's instructions using the primers and amplification conditions specified above. The 0.5 kb DIG-labeled probe was purified by 1 % agarose gel electrophoreseis using TAE buffer and excised from the gel and extracted using a MINELUTE® Gel Extraction Kit. The probe was boiled for 5 minutes, chilled on ice for 2 minutes, and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42°C for 15-17 hours. The membrane was then washed under low stringency conditions in 2X SSC plus 0.1 % SDS for 5 minutes at room temperature followed by two high stringency washes in 0.5X SSC plus 0.1 % SDS for 15 minutes each at 65°C. The probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Southern analysis indicated that all spore isolates contained the repair/replacement cassette at the ku70 locus and were cured of the hpt and tk markers. One strain designated T. reesei 981-O-8.5#10B+Ku70#19L3 was chosen for further transformations. Example 9: Construction of pDM286 expressing a Penicillium sp. GH61A polypeptide
The Penicillium sp. (emersonii) GH61A polypeptide coding sequence (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced amino acid sequence]) was amplified from plasmid pGH61 D23Y4 (WO 201 1/041397) using the gene-specific forward and reverse primers shown below. The region in italics represents vector homology to the site of insertion for an IN-FUSION® reaction.
Forward primer:
5'-CGG/\C7GCGC/\CCATGCTGTCTTCGACGACTCGCAC-3' (SEQ ID NO: 67)
Reverse primer:
5'-7CGCC/ACGG/\GCTTATCGACTTCTTCTAGAACGTC-3' (SEQ ID NO: 68)
The amplification reaction was composed of 30 ng of pGH61 D23Y4 DNA, 50 pmoles of each of the primers listed above, 1 μΙ of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, 1X PHUSION™ High-Fidelity Hot Start DNA Polymerase Buffer, and 1 unit of PHUSION™ High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 35 cycles each at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle at 72°C for 10 minutes. The PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where an approximately 0.9 kb fragment was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's protocol.
Plasmid pMJ09 (WO 2005/047499) was digested with Nco I and Pac I, isolated by 1.0% agarose gel electrophoresis in 1 mM disodium EDTA-50 mM Tris base-50 mM boric acid (TBE) buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
The 0.9 kb PCR product was inserted into the gel-purified Nco \IPac I digested pMJ09 using an IN-FUSION™ Advantage PCR Cloning Kit according to the manufacturer's protocol. The IN-FUSION™ reaction was composed of 1X IN-FUSION™ Reaction Buffer, 180 ng of the gel-purified Nco \IPac I digested pMJ09, 108 ng of the 0.9 kb PCR product, and 1 μΙ of IN-FUSION™ Enzyme in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 37°C and 15 minutes at 50°C. After the incubation period 40 μΙ of TE were added to the reaction. A 2 μΙ aliquot was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The E. coli transformation reactions were spread onto 2XYT plus ampicillin plates. The transformants were screened by sequencing and one clone containing the insert with no PCR errors was identified and designated pDM286 (Figure 4). Plasmid pDM286 can be digested with Pme I to generate an approximately 5.4 kb fragment for T. reesei transformation. The 5.4 kb fragment contains the expression cassette composed of the T. reesei Cel7A cellobiohydrolase I gene promoter, P. emersonii GH61A polypeptide coding sequence, and T. reesei Cel7A cellobiohydrolase I gene terminator. The 5.4 kb fragment also contains the Aspergillus nidulans acetamidase {amdS) gene.
Example 10: Generation of a Trichoderma reesei expression vector encoding Aspergillus fumigatus beta-glucosidase (Cel3A) mutant gene
A variant of the Aspergillus fumigatus Family 3A beta-glucosidase containing the substitutions G142S, Q183R, H266Q, and D703G was constructed by performing site- directed mutagenesis on pEJG97 (WO 2005/074647) using a QUIKCHANGE® Multi Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). A summary of the oligos used for the site-directed mutagenesis are shown in Table 1.
The resulting variant plasmid pDFng128-6 was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, CA, USA). The variant plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) to verify the changes.
TABLE 1
Figure imgf000078_0001
acid Plasmid changes Name
ccctttgggtatccgtGACtgtgagctatacccgcg
F100D AfBGmutFI OODF
(SEQ ID NO: 69)
cgtcatgagtgactggGGCgctcaccacagcggtg
S283G AfBGmutS283GF
(SEQ ID NO: 70) pDFng128- gggtagtggtactgccGAGttcccttaccttgtcac 6
N456E AfBGmutN456EF
(SEQ ID NO: 71 )
gccgactctggagagggtTACatcagtgtcgacggcaac
F512Y AfBGmutF512YF
(SEQ ID NO: 72)
Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-glucosidase mutant coding sequence from plasmid pDFng128-6. An IN-FUSION™ Cloning Kit was used to clone the fragment directly into the expression vector pMJ09. Bold letters represent coding sequence. The remaining sequence is homologous to insertion sites of pMJ09.
Forward primer:
5'-CGGACTGCGCACCATGAGATTCGGTTGGCTCGA-3' (SEQ ID NO: 73)
Reverse primer:
5'-TCGCCACGGAGCTTACTAGTAGACACGGGGCAGAG-3' (SEQ ID NO: 74)
Fifty picomoles of each of the primers above were used in a PCR reaction composed of 50 ng of pDFng128-6, 1X EXPAND® High Fidelity PCR Buffer with MgCI2 (Roche Diagnostics Corporation, Indianapolis, IN, USA), 0.25 mM each of dATP, dTTP, dGTP, and dCTP, and 2.6 units of EXPAND® High Fidelity Enzyme Mix (Roche Diagnostics Corporation, Indianapolis, IN, USA) in a final volume of 50 μΙ. The amplification was performed in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 94°C for 2 minute; 30 cycles each at 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 1 minute; and a final elongation at 68°C for 7 minutes. The heat block then went to a 4°C soak cycle. The reaction products were isolated by 0.7% agarose gel electrophoresis in TBE buffer where an approximately 3.1 kb product band was observed on the gel. The PCR reaction was purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
Plasmid pMJ09 was digested with Nco I and Pac I, isolated by 1 .0% agarose gel electrophoresis in TBE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The 3.1 kb gene fragment and the digested vector were ligated together using an I NFUSION™ Cloning Kit resulting in pDFng1 13-3 (Figure 5) in which transcription of the beta- glucosidase mutant coding sequence was under the control of a promoter from the Trichoderma reesei cbhl gene. The ligation reaction (20 μΙ) was composed of 1 X IN- FUSION™ Buffer, 1X BSA, 1 μΙ of IN-FUSION™ Enzyme (diluted 1 :10), 200 ng of the gel- purified Nco \IPac I digested pMJ09, and 172.2 ng of the purified 3.1 kb PCR product. The reaction was incubated at 37°C for 15 minutes followed by 50°C for 15 minutes. Two μΙ of the reaction was used to transform £ coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, CA, USA). The £ coli transformation reactions were spread onto 2XYT plus ampicillin plates. An £. coli transformant containing pDFng133-3 was prepared using a BIOROBOT® 9600. The Aspergillus fumigatus beta-glucosidase mutant insert in pDFng133 was confirmed by DNA sequencing.
Example 11 : Construction of plasmid pSMai139
To construct pSMai139, the Humicola insolens endoglucanase V full-length coding region was PCR amplified from pMJ05 (US 2004/0248258 A1 ) as template with the primers shown below. The underlined portions are Sph I and a Hind III sites introduced by the Car- F2 sense primer. The bold portion is an Eco Rl site introduced by the Car-R2 antisense primer.
Car-F2 sense primer:
5'-TATAAGCTTAAGCATGCGTTCCTCCCCCCTC-3' (SEQ ID NO: 75)
Car-R2 antisense primer:
5'-CTG CAG AATTCTACAG G CACTG ATG GTACCAG-3 ' (SEQ ID NO: 76)
The amplification reactions (50 μΙ) were composed of 1X ThermoPol Reaction Buffer (New England Biolabs, Inc., Ipswich, MA USA), 0.3 mM dNTPs, 10 ng of pMJ05 DNA, 0.3 μΜ Car-F2 sense primer, 0.3 μΜ Car-R2 antisense primer, and 2.5 units of VENT® DNA polymerase (New England Biolabs, Inc., Ipswich, MA USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 30 cycles each at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 900 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The 900 bp PCR fragment was then digested with Eco Rl and Hind III and subjected to a QIAQUICK® PCR Purification Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol. Plasmid pMJ05 was digested with Eco Rl and Hind III, isolated by 0.7% agarose gel electrophoresis in TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
The 900 bp Eco Rl and Hind III digested PCR fragment was ligated using T4 DNA ligase (Roche, Indianapolis, IN, USA) into Eco Rl and Hind III digested pMJ05. The ligation reaction was composed of 50 ng of the Eco Rl and Hind III digested pMJ05, 33 ng of the Eco Rl and Hind III digested 0.9 kb PCR fragment, 1X Ligase Buffer (Roche, Indianapolis, IN, USA), and 2 units of T4 DNA ligase in a final volume of 20 μΙ. The reaction was incubated at 15°C for 17 hours and 2 μΙ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.The resulting transformants were screened by restriction digestion analysis with Sph I and Bam HI to determine the presence and orientation of the insert and positive clones were sequenced. A clone containing the Humicola insolens endoglucanase V coding region with no PCR errors was designated pSMai139 (Figure 6).
Example 12: Construction of pSMai143 plasmid
Plasmid pSMai143 was constructed by amplifying 620 bp of the Trichoderma reesei cellobiohydrolase Cel6A promoter from Trichoderma reesei RutC30 genomic DNA using primers 994148 and 994149 shown below. The underlined portion is a Sal I site introduced by primer 994148. The bold portion is a "CAT" sequence introduced by primer 994149.
Primer 994148:
5'-ACGCGTCGACGAATTCTAGGCTAGGTATGCGAGGCA-3' (SEQ ID NO: 77)
Primer 994149:
5'-CATGGTGCAATACACAGAGGGTG-3' (SEQ ID NO: 78)
The amplification reactions (50 μΙ) were composed of 1X ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 0.3 μΜ 994148 sense primer, 0.3 μΜ 994149 antisense primer, and 2.5 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 30 cycles each at 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 620 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. Plasmid pSMai139 was digested with Sph I, 3'-protruding end blunted with T4 DNA polymerase and then digested with Sal I. The digested DNA was isolated by 0.7% agarose gel electrophoresis in TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
The 620 bp Sal I digested PCR fragment was ligated using T4 DNA ligase into Sph I and Sal I digested pSMai139. The ligation reaction was composed of 50 ng of the Sph I and Sal I digested pSMai139, 22 ng of the Sal I digested 0.62 kb PCR fragment, "IX Ligase Buffer, and 2 units of T4 DNA ligase in a final volume of 20 μΙ. The reaction was incubated at 15°C for 17 hours and 2 μΙ of the reaction was used to transform ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. The cells were heat shocked at 42°C for 30 seconds and 250 μΙ of SOC medium were added. The tubes were incubated at 37°C, 200 rpm for 1 hour and 250 μΙ were plated onto 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight.The resulting transformants were screened by restriction digestion analysis with Eco Rl to determine the presence and orientation of the insert and positive clones were sequenced. A clone containing the Trichoderma reesei cellobiohydrolase Cel6A promoter with no PCR errors was designated pSMai143 (Figure 7).
Example 13: Construction of plasmid pAG121
Expression vector pAG121 with an Nco I restriction site was constructed by performing site-directed mutagenesis on pSMai143 (Example 12) using a QUIKCHANGE® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using the primers shown below. The mutagenesis was performed according to manufacturer's recommendations using 20 ng of plasmid pAG121 and 12.5 μΜ primers in a final volume of 50 μΙ.
Smai143 SDM Fwd:
gtgtattgcaccatggcgttcctcccccctcc (SEQ ID NO: 79)
Smai143 SDM Rev
ggaggggggaggaacgccatggtgcaataca (SEQ ID NO: 80)
The resulting variant plasmid pAG121 was prepared using a BIOROBOT® 9600. The variant plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer to verify the changes.
Example 14: Construction of a Trichoderma reesei expression vector, pSMai229, encoding an Aspergillus fumigatus beta-glucosidase (Cel3A) mutant gene
A Trichoderma reesei expression vector, pSMai229, encoding the Aspergillus fumigatus beta-glucosidase (Cel3A) mutant coding sequence of Example 9, was constructed from pDFng133-3 (Example 10) and pAG121 (Example 13). The Aspergillus fumigatus beta-glucosidase (Cel3A) mutant coding sequence was PCR amplified from pDFng133-3 using primers 061 1689 and 061 1690 shown below. The regions in bold represent pAG121 vector homology to the site of insertion for IN-FUSION® cloning.
Primer 061 1689:
CACCCTCTGTGTATTGCACCATGAGATTCGGTTGGCTCGA (SEQ ID NO: 81 )
Primer 061 1690:
TTCGCCACGGAGCTACTAGTCTAGTAGACACGGGGCAGAG (SEQ ID NO: 82)
The amplification reaction was composed of 25 ng of pDFng133-3 DNA, 200 μηη dNTP's, 0.4 μΜ primers, 1X PHUSION® Buffer, and 1 unit of PHUSION® Hot Start High Fidelity DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 30 seconds, 56°C for 30 seconds, and 72°C for 3 minutes 30 seconds; and 1 cycle at 72°C for 15 minutes.
PCR products were separated by 1 % agarose gel electrophoresis using TAE buffer where a 3100 bp fragment was excised from the gel and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions. The fragment was then cloned to the largest fragment of pAG121 digested with Nco\ and Spel using an IN-FUSION™ Advantage PCR Cloning Kit resulting in pSMai229 (Figure 8). The ligation reaction (10 μΙ) was composed of 1X IN-FUSION™ Buffer, 1 μΙ of IN-FUSION™ Enzyme, 100 ng of pAG121 digested with Nco I and Spe I, and 142 ng of 3100 bp purified PCR product. The reaction was incubated at 37°C for 15 minutes followed by 15 minutes at 50°C. After diluting the reaction mix with 50 μΙ of TE buffer (pH 8), 2.5 μΙ of the reaction was used to transform £. coli ONE SHOT® TOP10 competent cells according to the manufacturer's protocol. An £. coli transformant containing pSMai229 was detected by restriction digestion and plasmid DNA was prepared using a BIOROBOT® 9600. The Aspergillus fumigatus beta-glucosidase (Cel3A) mutant insert in pSMai229 was confirmed by DNA sequencing.
Example 15: Co-transformation of pDM286 and pSMai229 into Trichoderma reesei 981 - O-8.5#10B+Ku70#19L3
Protoplast preparation and transformation of Trichoderma reesei strain 981-0- 8.5#10B+Ku70#19L3 was performed as described in Example 2.
Approximately 100 μg of pDM286 and pSMai229 were digested with Pme I. Each digestion reaction was purified by 1 % agarose gel electrophoresis in TAE buffer, a DNA band was excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit. Transformation was performed by adding 0.7-1.7 μg of Pme I digested and gel-purified pSMai229 and 0.7-2.0 of pDM286 to 100 μΙ of Trichoderma reesei T. reesei 981 -0- 8#10B+Ku70#19L3 protoplast solution and mixed gently. PEG buffer (250 μΙ) was added, mixed, and incubated at 34°C for 30 minutes. STC (4 ml) was then added, mixed, and plated onto COVE plates. The plates were incubated at 28°C for 7-10 days. After a single round of spore purification on COVE2 plus 10 mM Uridine plates, 362 transformants were grown in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing medium for 5 days at 28°C with agitation at 200 rpm. Culture broth samples were removed 5 days post- inoculation, centrifuged at 2000 rpm for 20 minutes, and the supernatants transferred to new tubes and stored at -20°C until enzyme assay.
The supernatants were assayed for beta-glucosidase activity using p-nitrophenyl- beta-D-glucopyranoside as substrate. Briefly, culture supernatants were diluted appropriately in 0.1 M succinate-0.01 % TRITON® X-100 pH 5.0 buffer (sample buffer) followed by a series dilution from 0-fold to 1/3-fold to 1/9-fold of the diluted sample. T. reesei RutC30 fermentation broth was initially diluted 1/64 followed with 2-fold dilution steps down to a 16- fold dilution in the sample buffer to establish the assay linear range. A total of 20 μΙ of each dilution was transferred to a 96-well flat bottom plate. Two hundred microliters of a 1 mg/ml p-nitrophenyl-beta-D-glucopyraniside substrate in 0.1 M succinate pH 5.0 buffer was added to each well and then incubated at ambient temperature for 45 minutes. Upon completion of the incubation period 50 μΙ of quenching solution (1 M Tris pH 9 buffer) was added per well. An endpoint was measured at an optical density of 405 nm for the 96-well plate. Sample activity was determined according to the following equation: (((OD405/ec)*1x106)/incubation time)/sample volume, where ec=17,749, incubation time = 45 minutes, and sample volume = 0.02 ml.
A number of transformants showed beta-glucosidase activity several-fold higher than that of Trichoderma reesei 981-O-8.5#10B+Ku70#19L3. All samples with beta-glucosidase activity values greater than 7000 μΜ/min/ml were analyzed by SDS-PAGE using CRITERION® 8-16% Tris-HCI gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a CRITERION® Cell (Bio-Rad Laboratories, Inc. Hercules, CA, USA) to determine Penicillium emersonii GH61A polypeptide expression. Five μΙ of day 5 samples were suspended in 2X concentration of Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) and heated at 95°C for 5 minutes in the presence of 5% beta-mercaptoethanol. All samples were loaded onto the CRITERION® 8-16% Tris-HCI gels and subjected to electrophoresis in 1 X Tris/Glycine/SDS running buffer (Bio-Rad Laboratories, Hercules, CA, USA). The resulting gels were stained with BIO-SAFE® Coomassie Stain (Bio-Rad Laboratories, Hercules, CA, USA). SDS-PAGE profiles of the cultures showed the presence of both the Aspergillus fumigatus beta-glucosidase variant and the Penicillium emersonii GH61A in samples #1 , 64, 79, 82, 83, 1 16, 147, 167, 193, 198, 210, 219, 908, 922, 928, 930, 935, 951 , 963, and 980.
Example 16: Construction of pAG57
The Aspergillus fumigatus strain NN051616 GH3 beta-xylosidase (SEQ ID NO: 15
[DNA sequence] and SEQ ID NO: 16 [deduced amino acid sequence]) was recombinantly prepared according to the following procedure.
Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-xylosidase gene from genomic DNA. Genomic DNA was prepared as described in Example 1 . An I N-FUSION™ Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pAILo2 (WO 2005/074647), without the need for restriction digests and ligation.
Forward primer:
5'-ACTGGATTTACCATGGCGGTTGCCAAATCTATTGCT-3' (SEQ ID NO: 83)
Reverse primer:
5'-TCACCTCTAGTTAATTAATCACGCAGACGAAATCTGCT-3' (SEQ ID NO: 84)
Bold letters represent coding sequence. The remaining sequence is homologous to the insertion sites of pAILo2.
Fifteen picomoles of each of the primers above were used in a PCR reaction containing 250 ng of Aspergillus fumigatus genomic DNA, 1 X EXPAND® High Fidelity PCR Buffer with MgCI2, 1 μΙ of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, and 0.75 units of EXPAND® High Fidelity Enzyme Mix in a final volume of 50 μΙ. The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 94°C for 2 minutes; 10 cycles each at 94°C for 15 seconds, 56.5°C for 30 seconds, and 72°C for 2 minutes; and 20 cycles each at 94°C for 15 seconds, 56.5°C for 30 seconds, and 72°C for 2 minutes plus 5 seconds per successive cycle. The heat block was then held at 72°C for 7 minutes followed by a 4°C soak cycle.
The reaction products were isolated by 1 .0% agarose gel electrophoresis using TAE buffer where a 2.4 kb product band was excised from the gel and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions.
The fragment was then cloned into pAILo2 using an IN-FUSION™ Advantage PCR Cloning Kit. The vector was digested with Nco I and Pac I . The fragment was purified by 1 % agarose gel electrophoresis using TAE buffer, excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit. The gene fragment and the digested vector were combined together in a reaction resulting in the expression plasmid pAG57, in which transcription of the Aspergillus fumigatus beta-xylosidase coding sequence was under the control of the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase). The reaction (20 μΙ) was composed of 1X IN-FUSION™ Buffer, 1X BSA, 1 μΙ of IN-FUSION™ Enzyme (diluted 1 :10), 182 ng of pAILo2 digested with Nco I and Pac I, and 97.7 ng of the Aspergillus fumigatus beta-xylosidase purified PCR product. The reaction was incubated at 37°C for 15 minutes followed by 15 minutes at 50°C. The reaction was diluted with 40 μΙ of TE buffer and 2.5 μΙ of the diluted reaction were used to transform E. coli TOP10 Competent cells. An £. coli transformant containing pAG57 (Figure 9) was identified by restriction enzyme digestion and plasmid DNA was prepared using a BIOROBOT® 9600. The pAG57 plasmid construct was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer to verify the sequence.
Example 17: Construction of pDFng 124-1 expressing an Aspergillus fumigatus beta- xylosidase
Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus beta-xylosidase from pAG57 (Example 16). An IN-FUSION™ Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pMJ09, without the need for restriction digestion and ligation.
Forward primer:
5'-CGGACTGCGCACCATGGCGGTTGCCAAATC-3' (SEQ ID NO: 85)
Reverse primer:
5'-TCGCCACGGAGCTTATCACGCAGACGAAATCT-3' (SEQ ID NO: 86)
Bold letters represent coding sequence. The remaining sequence was homologous to insertion sites of pMJ09.
Fifty picomoles of each of the primers above were used in a PCR reaction composed of 100 ng of pAG57, 1X EXPAND® High Fidelity PCR buffer with MgCI2, 0.25 mM each of dATP, dTTP, dGTP, and dCTP, and 2.6 units of EXPAND® Enzyme Mix in a final volume of 50 μΙ. The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 94°C for 2 minutes; 30 cycles each at 94°C for 15 seconds, 65°C for 30 seconds, and 72°C for 2 minutes; and a final elongation at 72°C for 7 minutes. The heat block then went to a 4°C soak cycle.
The reaction products were isolated by 0.7% agarose gel electrophoresis in TBE buffer where a 2.4 kb product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
Plasmid pMJ09 was digested with Nco I and Pac I , isolated by 0.7% agarose gel electrophoresis in TBE buffer, and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The gene fragment and the digested vector were ligated together using an INFUSION™ Advantage PCR Cloning Kit resulting in pDFng124-1 (Figure 10) in which transcription of the beta-xylosidase coding sequence was under the control of the Trichoderma reesei cbhl gene promoter. The ligation reaction (20 μΙ) was composed of 1X IN-FUSION™ Buffer, 1 μΙ of IN-FUSION™ Enzyme (diluted 1 :10), 200 ng of pMJ09 digested with Nco I and Pac I, and 100 ng of the purified beta-xylosidase PCR product. The reaction was incubated at 37°C for 15 minutes followed by 50°C for 15 minutes. Two μΙ of the reaction were used to transform E. coli XL10 SOLOPACK® Gold Supercompetent cells according to manufacturer's instructions. An E. coli transformant containing pDFng124-1 was prepared using a BIOROBOT® 9600. The Aspergillus fumigatus beta-xylosidase insert in pDFng124-1 was confirmed by DNA sequencing.
Example 18: Construction pSaMe-AFGH10 expressing an Aspergillus fumigatus xylanase
Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Aspergillus fumigatus GH10 xylanase from pHyGe001 (WO 2006/078256). An INFUSION™ Advantage PCR Cloning Kit was used to clone the fragment directly into the expression vector, pMJ09, without the need for restriction digestion and ligation.
Forward primer:
5'-CGGACTGCGCACCATGGTCCATCTATCTTCATT-3' (SEQ ID NO: 87)
Reverse primer:
5'-TCGCCACGGAGCTTATTACAGGCACTGTGAGTACC-3' (SEQ ID NO: 88)
Bold letters represent coding sequence. The remaining sequence was homologous to the insertion sites of pMJ09.
Fifty picomoles of each of the primers above were used in a PCR reaction composed of 50 ng of pHYGE001 , 1 μΙ of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, 5 μΙ of 10X ACCUTAQ™ DNA Polymerase Buffer (Sigma-Aldrich, St. Louis, MO, USA), and 5 units of ACCUTAQ™ DNA Polymerase (Sigma-Aldrich, St. Louis, MO, USA) in a final volume of 50 μΙ. An EPPENDORF® MASTERCYCLER® 5333 epgradient S was used to amplify the DNA fragment programmed for 1 cycle at 95°C for 2 minutes; and 30 cycles each at 94°C for 15 seconds, 55°C for 30 seconds, and 68°C for 1 minute. After the 30 cycles, the reaction was incubated at 72°C for 10 minutes and then cooled to 4°C until further processing.
The reaction products were isolated by 1.0% agarose gel electrophoresis using TAE buffer where a 1.4 kb product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.
The 1.4 kb fragment was then cloned into pMJ09 using an I N-FUSION™ Cloning Kit. Plasmid pMJ09 was digested with Nco I and Pac I and purified by agarose gel electrophoresis as described above. The gene fragment and the digested vector were ligated together in a reaction resulting in the expression plasmid pSaMe-AfGH10 in which transcription of the xylanase coding sequence was under the control of the T. reesei cbhl gene promoter. The ligation reaction (50 μΙ) was composed of 1X IN-FUSION™ Buffer, 1X BSA, 1 μΙ of IN-FUSION™ enzyme (diluted 1 :10), 100 ng of pMJ09 digested with Nco I and Pac I, and 100 ng of the Aspergillus fumigatus xylanase purified PCR product. The reaction was incubated at room temperature for 30 minutes. One μΙ of the reaction was used to transform E. coli XL10 SOLOPACK® Gold cells. An E. coli transformant containing pSaMe- AfGH10 (Figure 1 1 ) was detected by restriction enzyme digestion and plasmid DNA was prepared using a BIOROBOT® 9600. DNA sequencing of the Aspergillus fumigatus xylanase coding sequence from pSaMe-AfGH10 was performed using dye-terminator chemistry (Giesecke et al. , 1992, Journal of Virology Methods 38: 47-60) and primer walking strategy. Example 19: Generation of Trichoderma reesei RutC30 strain expressing Aspergillus fumigatus xylanase and Aspergillus fumigatus beta-xylosidase
Protoplast preparation and transformation of Trichoderma reesei strain RutC30 was performed as described in Example 2.
Approximately 100 μg of pSaMe-AFGH10 and pDFng124-1 were digested with Pme I. Each digestion reaction was purified by 0.65% agarose gel electrophoresis in TAE buffer, a DNA band was excised from the gel, and extracted using a QIAQUICK® Gel Extraction Kit. Transformation was performed by adding 2 μg of Pme I digested and gel-purified pDFng124- 1 and 1.72 μg of pSaMe-AfGH10 to 100 μΙ of Trichoderma reesei strain RutC30 protoplast solution and mixed gently. PEG buffer (250 μΙ) was added, mixed, and incubated at 34°C for 30 minutes. STC (6 ml) was then added, mixed, and plated onto COVE plates. The plates were incubated at 28°C for 7-10 days. After a single round of spore purification on COVE2 plus 10 mM uridine plates, 200 transformants were grown in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing medium for 5 days at 28°C with agitation at 200 rpm. Culture broth samples were removed 5 days post-inoculation, centrifuged at 2000 rpm for 20 minutes, and the supernatants transferred to new tubes and stored at -20°C until enzyme assay.
Three to five μΙ of each supernatant were combined with 5 to 6 μΙ of Laemelli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) with 5% beta-mercaptoethanol in a 0.2 ml microcentrifuge tube and boiled for 2 minutes at 95°C in an EPPENDORF® MASTERCYCLER® 5333 epgradient S. Samples were analyzed by SDS-PAGE using a CRITERION® 8-16% Tris-HCI Gel according to the manufacturer's instructions and 10 μΙ of PRECISION PLUS™ All Blue Protein Standards (Bio-Rad Laboratories, Hercules, CA, USA). Gels were stained with BIO-SAFE® Coomassie Stain.
Four strains were selected based on high expression of beta-xylosidase and xylanase polypeptide and were spore purified by adding spores collected on a 10 μΙ inoculation loop to 1.5 ml of 0.01 % TWEEN® 20. Spore dilutions of 1 :1500 and 1 :150 were spread onto 150 mm COVE plates and cultured for 4 days at 28°C. Four spore isolates per strain (total of 16 isolates) were obtained and transferred to COVE2 + 10 mM uridine plates and cultivated at 28°C for 9 days. The shake flask and SDS-PAGE procedures were repeated for the first round spore isolates. Eight strains were selected based on high expression of beta-xylosidase and xylanase polypeptide and were spore purified a second time as described above resulting in four spore isolates per strain (total of 32 isolates). The shake flask and SDS-PAGE procedures were repeated for the second round spore isolates. The final strain was selected based on high expression of beta-xylosidase and xylanase polypeptide and designated 06HY4.
Example 20: Pretreated corn stover hydrolysis assay
Corn stover was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1 .4 wt % sulfuric acid at 165°C and 107 psi for 8 minutes. The water-insoluble solids in the pretreated corn stover (PCS) contained 56.5% cellulose, 4.6% hemicelluloses, and 28.4% lignin. Cellulose and hemicellulose were determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003.
Milled unwashed PCS was prepared by milling whole slurry PCS in a Cosmos ICMG
40 wet multi-utility grinder (EssEmm Corporation, Tamil Nadu, India).
The hydrolysis of PCS was conducted using 2.2 ml deep-well plates (Axygen, Union City, CA, USA) in a total reaction volume of 1 .0 ml. The hydrolysis was performed with 50 mg of insoluble PCS solids per ml of 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate and various protein loadings of various enzyme compositions (expressed as mg protein per gram of cellulose). Enzyme compositions were prepared and then added simultaneously to all wells in a volume ranging from 50 μΙ to 200 μΙ, for a final volume of 1 ml in each reaction. The plates were then sealed using an ALPS-300™ plate heat sealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and incubated at a specific temperature for 72 hours. All experiments reported were performed in triplicate.
Following hydrolysis, samples were filtered using a 0.45 μηη MULTISCREEN® 96- well filter plate (Millipore, Bedford, MA, USA) and filtrates analyzed for sugar content as described below. When not used immediately, filtered aliquots were frozen at -20°C. The sugar concentrations of samples diluted in 0.005 M H2S04 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.05% w/w benzoic acid-0.005 M H2S04 at 65°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION®, AGI LENT® 1 100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. The resultant glucose and cellobiose equivalents were used to calculate the percentage of cellulose conversion for each reaction.
Glucose, cellobiose, and xylose were measured individually. Measured sugar concentrations were adjusted for the appropriate dilution factor. In case of unwashed PCS, the net concentrations of enzymatically-produced sugars were determined by adjusting the measured sugar concentrations for corresponding background sugar concentrations in unwashed PCS at zero time points. All HPLC data processing was performed using MICROSOFT EXCEL™ software (Microsoft, Richland, WA, USA).
The degree of cellulose conversion to glucose was calculated using the following equation: % conversion = (glucose concentration / glucose concentration limit digest) x 100. To calculate total conversion the glucose and cellobiose values were combined. The degree of total cellulose conversion was calculated using the following equation: % conversion = [glucose concentration] / [(glucose concentration in a limit digest] x 100. In order to calculate % conversion, a 100% conversion point was set based on a cellulase control (50 mg of Trichoderma reesei cellulase per gram cellulose), and all values were divided by this number and then multiplied by 100. Triplicate data points were averaged and standard deviation was calculated.
An enzyme composition comprising an Aspergillus fumigatus cellobiohydrolase I; an Aspergillus fumigatus cellobiohydrolase II; an Aspergillus fumigatus beta-glucosidase variant; a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity, an Aspergillus fumigatus xylanase, and an Aspergillus fumigatus beta-xylosidase (designated "enzyme composition #1 ") was compared to an enzyme composition comprising a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) (designated "enzyme composition #2").
Upon completion of the hydrolysis assay, a Protein Loading (mg EP/g cellulose) versus Percent Converison (%) graph was generated. Using linear interpolation the protein loading required to reach a certain percent conversion can be determined. In this case, 80% conversion of glucan to glucose equivalents was chosen to determine relative improvements of enzyme composition 1 as compared to enzyme composition 2. The results of this assay as shown in Figure 12 indicated that enzyme composition 1 is able to reach 80% conversion with 4.1 mg EP/g cellulose, whereas enzyme compositoni 2 is able to reach the same conversion target with 7.3 mg EP/g cellulose. This represents a 1.78 fold improvement in performance per milligram of protein for enzyme composition 1 over enzyme composition 2 or a 1.78 fold reduction in protein requirement to reach 80% conversion.
The present invention is further described by the following numbered paragraphs:
[1 ] An enzyme composition, comprising: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
[2] The enzyme composition of paragraph 1 , wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and (iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 or the full-length complement thereof.
[3] The enzyme composition of paragraph 1 , wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 or the full-length complement thereof.
[4] The enzyme composition of paragraph 1 , wherein the Aspergillus fumigatus beta- glucosidase or homolog thereof is selected from the group consisting of: (i) a beta- glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta- glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6; (iii) a beta- glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5; and (iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 or the full-length complement thereof.
[5] The enzyme composition of paragraph 1 , wherein the beta-glucosidase variant comprises a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 6, wherein the variant has beta- glucosidase activity.
[6] The enzyme composition of paragraph 5, wherein the parent beta-glucosidase of the variant is (a) a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 6; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or (e) a fragment of the mature polypeptide of SEQ ID NO: 6, which has beta-glucosidase activity.
[7] The enzyme composition of paragraph 5 or 6, wherein the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent beta- glucosidase.
[8] The enzyme composition of any of paragraphs 5-7, wherein the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 6.
[9] The enzyme composition of any of paragraphs 5-8, wherein the number of substitutions is 1-4, such as 1 , 2, 3, or 4 substitutions.
[10] The enzyme composition of any of paragraphs 5-9, wherein the variant comprises a substitution at a position corresponding to position 100, a substitution at a position corresponding to position 283, a substitution at a position corresponding to position 456, and/or a substitution at a position corresponding to position 512.
[1 1] The enzyme composition of paragraph 10, wherein the substitution at the position corresponding to position 100 is Ser; the substitution at the position corresponding to position 456 is Gly; the substitution at the position corresponding to position 456 is Gin; and the substitution at the position corresponding to position 512 is Gly.
[12] The enzyme composition of any of paragraphs 5-1 1 , wherein the variant comprises one or more (several) substitutions selected from the group consisting of G142S, Q183R, H266Q, and D703G.
[13] The enzyme composition of any of paragraphs 5-12, wherein the variant comprises the substitutions G142S and Q183R; G142S and H266Q; G142S and D703G; Q183R and H266Q; Q183R and D703G; H266Q and D703G; G142S, Q183R, and H266Q; G142S, Q183R, and D703G; G142S, H266Q, and D703G; Q183R, H266Q, and D703G; or G142S, Q183R, H266Q, and D703G.
[14] The enzyme composition of paragraph 1 , wherein the Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of: (i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and (iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 or the full-length complement thereof.
[15] The enzyme composition of any of paragraphs 1-14, which further comprises one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase or homolog thereof, (ii) an Aspergillus fumigatus beta-xylosidase or homolog thereof; or (iii) a combination of (i) and (ii).
[16] The enzyme composition of paragraph 15, wherein the Aspergillus fumigatus xylanase or homolog thereof is selected from the group consisting of: (i) an Aspergillus fumigatus xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ I D NO: 12, or SEQ ID NO: 14; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; and (iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ I D NO: 13; or the full-length complement thereof. [17] The enzyme composition of paragraph 15, wherein the Aspergillus fumigatus beta-xylosidase or homolog thereof is selected from the group consisting of: (i) a beta- xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16; (ii) a beta- xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 16; (iii) a beta- xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 15; and (iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 15 or the full-length complement thereof.
[18] The enzyme composition of any of paragraphs 1-17, which further comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[19] The enzyme composition of paragraph 18, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[20] The enzyme composition of paragraph 19, wherein the endoglucanase is an endoglucanase I.
[21] The enzyme composition of paragraph 19, wherein the endoglucanase is an endoglucanase II.
[22] The enzyme composition of paragraph 18, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[23] A recombinant filamentous fungal host cell, comprising polynucleotides encoding: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof. [24] The recombinant filamentous fungal host cell of paragraph 23, wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and (iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 or the full-length complement thereof.
[25] The recombinant filamentous fungal host cell of paragraph 23, wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of: (i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 or the full-length complement thereof.
[26] The recombinant filamentous fungal host cell of paragraph 23, wherein the
Aspergillus fumigatus beta-glucosidase or homolog thereof is selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6; (iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5; and (iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 or the full-length complement thereof.
[27] The recombinant filamentous fungal host cell of paragraph 23, wherein the beta- glucosidase variant comprises a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 6, wherein the variant has beta-glucosidase activity.
[28] The recombinant filamentous fungal host cell of paragraph 27, wherein the parent beta-glucosidase of the variant is (a) a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 6; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or (e) a fragment of the mature polypeptide of SEQ ID NO: 6, which has beta-glucosidase activity.
[29] The recombinant filamentous fungal host cell of paragraph 27 or 28, wherein the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent beta-glucosidase.
[30] The recombinant filamentous fungal host cell of any of paragraphs 27-29, wherein the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 6.
[31] The recombinant filamentous fungal host cell of any of paragraphs 27-30, wherein the number of substitutions is 1-4, such as 1 , 2, 3, or 4 substitutions.
[32] The recombinant filamentous fungal host cell of any of paragraphs 27-31 , wherein the variant comprises a substitution at a position corresponding to position 100, a substitution at a position corresponding to position 283, a substitution at a position corresponding to position 456, and/or a substitution at a position corresponding to position 512.
[33] The recombinant filamentous fungal host cell of paragraph 32, wherein the substitution at the position corresponding to position 100 is Ser; the substitution at the position corresponding to position 456 is Gly; the substitution at the position corresponding to position 456 is Gin; and the substitution at the position corresponding to position 512 is Gly.
[34] The recombinant filamentous fungal host cell of any of paragraphs 27-33, wherein the variant comprises one or more (several) substitutions selected from the group consisting of G142S, Q183R, H266Q, and D703G.
[35] The recombinant filamentous fungal host cell of any of paragraphs 27-34, wherein the variant comprises the substitutions G142S and Q183R; G142S and H266Q; G142S and D703G; Q183R and H266Q; Q183R and D703G; H266Q and D703G; G142S, Q183R, and H266Q; G142S, Q183R, and D703G; G142S, H266Q, and D703G; Q183R, H266Q, and D703G; or G142S, Q183R, H266Q, and D703G.
[36] The recombinant filamentous fungal host cell of paragraph 23, wherein the
Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of: (i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 8; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and (iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 or the full-length complement thereof.
[37] The recombinant filamentous fungal host cell of any of paragraphs 23-36, which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase; (ii) an Aspergillus fumigatus beta-xylosidase; and (iii) a combination of (i) and (ii).
[38] The recombinant filamentous fungal host cell of paragraph 37, wherein the Aspergillus fumigatus xylanase or homolog thereof is selected from the group consisting of: (i) an Aspergillus fumigatus xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; and (iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; or the full-length complement thereof.
[39] The recombinant filamentous fungal host cell of paragraph 37, wherein the Aspergillus fumigatus beta-xylosidase or homolog thereof is selected from the group consisting of: (i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16; (ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 16; (iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ I D NO: 15; and (iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 15 or the full-length complement thereof.
[40] The recombinant filamentous fungal host cell of any of paragraphs 23-39, which is a Trichoderma cell.
[41] The recombinant filamentous fungal host cell of paragraph 40, wherein the Trichoderma cell is selected from the group consisting of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.
[42] The recombinant filamentous fungal host cell of paragraph 40, which is
Trichoderma reesei.
[43] The recombinant filamentous fungal host cell of any of paragraphs 23-42, wherein one or more of the cellulase genes, one or more of hemicellulase genes, or a combination thereof, endogenous to the filamentous fungal host cell have been inactivated.
[44] The recombinant filamentous fungal host cell of paragraph 43, wherein a cellobiohydrolase I gene has been inactivated.
[45] The recombinant filamentous fungal host cell of paragraph 44, wherein the cellobiohydrolase I gene encodes a cellobiohydrolase I selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 18; (ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 18; (iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 17; and (iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 17 or the full-length complement thereof.
[46] The recombinant filamentous fungal host cell of any of paragraphs 43-45, wherein a cellobiohydrolase II gene has been inactivated.
[47] The recombinant filamentous fungal host cell of paragraph 46, wherein the cellobiohydrolase II gene encodes a cellobiohydrolase II selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 20; (ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 20; (iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 19; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 19 or the full-length complement thereof.
[48] The recombinant filamentous fungal host cell of any of paragraphs 43-47, wherein a beta-glucosidase gene has been inactivated.
[49] The recombinant filamentous fungal host cell of paragraph 48, wherein the beta- glucosidase gene encodes a beta-glucosidase selected from the group consisting of: (i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 22; (ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 22; (iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 21 ; and (iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 21 or the full-length complement thereof.
[50] The recombinant filamentous fungal host cell of any of paragraphs 43-49, wherein a xylanase I gene has been inactivated.
[51] The recombinant filamentous fungal host cell of paragraph 50, wherein the xylanase I gene encodes a xylanase I selected from the group consisting of: (i) a xylanase I comprising or consisting of the mature polypeptide of SEQ ID NO: 24; (ii) a xylanase I comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 24; (iii) a xylanase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 23; and (iv) a xylanase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 23 or the full-length complement thereof.
[52] The recombinant filamentous fungal host cell of any of paragraphs 43-51 , wherein a xylanase II gene has been inactivated.
[53] The recombinant filamentous fungal host cell of paragraph 52, wherein the xylanase II gene encodes a xylanase II selected from the group consisting of: (i) a xylanase II comprising or consisting of the mature polypeptide of SEQ ID NO: 26; (ii) a xylanase II comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 26; (iii) a xylanase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25; and (iv) a xylanase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 25 or the full-length complement thereof.
[54] The recombinant filamentous fungal host cell of any of paragraphs 43-53, wherein a Trichoderma reesei xylanase III gene has been inactivated.
[55] The recombinant filamentous fungal host cell of paragraph 54, wherein the xylanase III gene encodes a xylanase III selected from the group consisting of: (i) a xylanase III comprising or consisting of the mature polypeptide of SEQ ID NO: 28; (ii) a xylanase III comprising or consisting of an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 28; (iii) a xylanase III encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 27; and (iv) a xylanase III encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 27 or the full-length complement thereof.
[56] The recombinant filamentous fungal host cell of any of paragraphs 43-55, wherein a beta-xylosidase gene has been inactivated.
[57] The recombinant filamentous fungal host cell of paragraph 56, wherein the beta- xylosidase gene encodes a beta-xylosidase selected from the group consisting of: (i) a beta- xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 30 (ii) a beta- xylosidase comprising or consisting of an amino acid sequence having at least 70%, e.g. , at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 30; (iii) a beta- xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29; and (iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g. , very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 29 or the full-length complement thereof.
[58] The recombinant filamentous fungal host cell of any of paragraphs 23-55, which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[59] The recombinant filamentous fungal host cell of paragraph 58, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[60] The recombinant filamentous fungal host cell of paragraph 58, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[61 ] The recombinant filamentous fungal host cell of any of paragraphs 58-60, wherein one or more of the enzymes are native to the filamentous fungal host cell.
[62] The recombinant filamentous fungal host cell of paragraph 61 , wherein the enzyme native to the filamentous fungal host cell is an endoglucanase.
[63] The recombinant filamentous fungal host cell of paragraph 62, wherein the endoglucanase is an endoglucanase I.
[64] The recombinant filamentous fungal host cell of paragraph 62, wherein the endoglucanase is an endoglucanase II.
[65] A method of producing an enzyme composition, comprising: (a) cultivating the host cell of any of paragraphs 23-64 under conditions conducive for production of the enzyme composition; and optionally (b) recovering the enzyme composition.
[66] A process for degrading a cellulosic material, comprising: treating the cellulosic material with the enzyme composition of any of paragraphs 1-22.
[67] The process of paragraph 66, wherein the cellulosic material is pretreated.
[68] The process of paragraph 66 or 67, further comprising recovering the degraded cellulosic material.
[69] The process of paragraph 68, wherein the degraded cellulosic material is a sugar
[70] The process of paragraph 69, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.
[71 ] A process for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with the enzyme composition of any of paragraphs 1-22; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[72] The process of paragraph 71 , wherein the cellulosic material is pretreated.
[73] The process of paragraph 71 or 72, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.
[74] The process of any of paragraphs 71-73, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.
[75] A process of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with the enzyme composition of any of paragraphs 1 -22.
[76] The process of paragraph 75, wherein the fermenting of the cellulosic material produces a fermentation product.
[77] The process of paragraph 76, further comprising recovering the fermentation product from the fermentation.
[78] The process of paragraph 76 or 77, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.
[79] The process of any of paragraphs 75-78, wherein the cellulosic material is pretreated before saccharification.
[80] The enzyme composition of paragraphs 47-49, further comprising a Trichoderma endoglucanase I, a Trichoderma endoglucanase II, or a Trichoderma endoglucanase I and a Trichoderma endoglucanase II .
[81] The enzyme composition of paragraph 80, wherein the Trichoderma endoglucanase I is a Trichoderma reesei endoglucanase I.
[82] The enzyme composition of paragraph 80, wherein the Trichoderma endoglucanase II is a Trichoderma reesei endoglucanase II.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

Claims
What is claimed is: 1 . An enzyme composition, comprising: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta- glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
2. The enzyme composition of claim 1 , wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2;
(ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2;
(iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and
(iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 or the full-length complement thereof;
wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 4;
(ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4;
(iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and
(iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 or the full-length complement thereof;
wherein the Aspergillus fumigatus beta-glucosidase or homolog thereof is selected from the group consisting of:
(i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6;
(ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6;
(iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5;
(iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 or the full-length complement thereof; and
(v) a beta-glucosidase variant comprising a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ I D NO: 6, wherein the variant has beta-glucosidase activity; and
wherein the Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of:
(i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 6; (ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8;
(iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and
(iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 or the full-length complement thereof.
3. The enzyme composition of claim 1 or 2, wherein the beta-glucosidase variant comprises one or more (several) substitutions selected from the group consisting of G142S,
Q183R, H266Q, and D703G.
4. The enzyme composition of any of claims 1 -3, which further comprises one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase or homolog thereof, (ii) an Aspergillus fumigatus beta-xylosidase or homolog thereof; or (iii) a combination of (i) and (ii);
wherein the Aspergillus fumigatus xylanase or homolog thereof is selected from the group consisting of:
(i) an Aspergillus fumigatus xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;
(ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; and
(iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; or the full-length complement thereof; and
wherein the Aspergillus fumigatus beta-xylosidase or homolog thereof is selected from the group consisting of:
(i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16;
(ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 16;
(iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 15; and
(iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 15 or the full-length complement thereof.
5. The enzyme composition of any of claims 1 -4, which further comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
6. A recombinant filamentous fungal host cell, comprising polynucleotides encoding: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
7. The recombinant filamentous fungal host cell of claim 6, wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 2;
(ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2;
(iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and
(iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 or the full-length complement thereof;
wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of
SEQ ID NO: 4;
(ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 4;
(iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and
(iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 or the full-length complement thereof;
wherein the Aspergillus fumigatus beta-glucosidase or homolog thereof is selected from the group consisting of:
(i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 6;
(ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 6;
(iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5;
(iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 or the full-length complement thereof; and
(v) beta-glucosidase variant comprising a substitution at one or more positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO:
6, wherein the variant has beta-glucosidase activity; and
wherein the Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of:
(i) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of the mature polypeptide of SEQ ID NO: 8;
(ii) a GH61 polypeptide having cellulolytic enhancing activity comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 8; (iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7; and
(iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 or the full-length complement thereof.
8. The recombinant filamentous fungal host cell of claim 6 or 7, wherein the beta- glucosidase variant comprises one or more (several) substitutions selected from the group consisting of G142S, Q183R, H266Q, and D703G.
9. The recombinant filamentous fungal host cell of any of claims 6-8, which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of: (i) an Aspergillus fumigatus xylanase; (ii) an Aspergillus fumigatus beta- xylosidase; and (iii) a combination of (i) and (ii);
wherein the Aspergillus fumigatus xylanase or homolog thereof is selected from the group consisting of:
(i) an Aspergillus fumigatus xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;
(ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:;
(iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; and (iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 9, SEQ ID NO: 1 1 , or SEQ ID NO: 13; or the full-length complement thereof; and
wherein the Aspergillus fumigatus beta-xylosidase or homolog thereof is selected from the group consisting of:
(i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 16;
(ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 16;
(iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 15; and
(iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 15 or the full-length complement thereof.
10. The recombinant filamentous fungal host cell of any of claims 6-9, which is a Trichoderma cell.
1 1 . The recombinant filamentous fungal host cell of claim 10, which is Trichoderma reesei.
12. The recombinant filamentous fungal host cell of any of claims 6-1 1 , wherein one or more cellulase genes, one or more hemicellulase genes, or a combination thereof, endogenous to the filamentous fungal host cell have been inactivated.
13. The recombinant filamentous fungal host cell of claim 12, wherein the cellulase gene is a a cellobiohydrolase I gene that has been inactivated; wherein the cellobiohydrolase I gene encodes a cellobiohydrolase I selected from the group consisting of: (i) a cellobiohydrolase I comprising or consisting of the mature polypeptide of SEQ ID NO: 18;
(ii) a cellobiohydrolase I comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 18;
(iii) a cellobiohydrolase I encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 17; and
(iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 17 or the full-length complement thereof.
14. The recombinant filamentous fungal host cell of claim 12 or 13, wherein the cellulase gene is a a cellobiohydrolase II gene that has been inactivated; wherein the cellobiohydrolase II gene encodes a cellobiohydrolase II selected from the group consisting of:
(i) a cellobiohydrolase II comprising or consisting of the mature polypeptide of SEQ ID NO: 20;
(ii) a cellobiohydrolase II comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 20;
(iii) a cellobiohydrolase II encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 19; and (iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 19 or the full-length complement thereof.
15. The recombinant filamentous fungal host cell of any of claims 12-14, wherein the cellulase gene is a beta-glucosidase gene that has been inactivated; wherein the beta- glucosidase gene encodes a beta-glucosidase selected from the group consisting of:
(i) a beta-glucosidase comprising or consisting of the mature polypeptide of SEQ ID NO: 22;
(ii) a beta-glucosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 22;
(iii) a beta-glucosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 21 ; and
(iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 21 or the full-length complement thereof.
16. The recombinant filamentous fungal host cell of any of claims 12-15, wherein the hemicellulase gene is a xylanase gene that has been inactivated; wherein the xylanase gene encodes a xylanase selected from the group consisting of:
(i) a xylanase comprising or consisting of the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28;
(ii) a xylanase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 2; (iii) a xylanase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27; and
(iv) a xylanase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27 or the full-length complement thereof.
17. The recombinant filamentous fungal host cell of any of claims 12-16, wherein the hemicellulase gene is a beta-xylosidase gene that has been inactivated; wherein the beta- xylosidase gene encodes a beta-xylosidase selected from the group consisting of:
(i) a beta-xylosidase comprising or consisting of the mature polypeptide of SEQ
ID NO: 30
(ii) a beta-xylosidase comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 30;
(iii) a beta-xylosidase encoded by a polynucleotide comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29; and
(iv) a beta-xylosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 29 or the full-length complement thereof.
18. The recombinant filamentous fungal host cell of any of claims 6-17, which further comprises one or more polynucleotides encoding one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
19. A method of producing an enzyme composition, comprising: cultivating the host cell of any of claims 7-18 under conditions conducive for production of the enzyme composition.
20. The method of claim 19, further comprising recovering the enzyme composition.
21 . A process for degrading a cellulosic material, comprising: treating the cellulosic material with the enzyme composition of any of claims 1 -6.
22. A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with the enzyme composition of any of claims 1-6;
(b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
23. A process of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with the enzyme composition of any of claims 1 -6.
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BR112014004186-5A BR112014004186B1 (en) 2011-08-24 2012-08-23 ENZYMATIC COMPOSITION, RECOMBINANT FILAMENT FUNGUS HOSTING CELL, METHOD TO PRODUCE AN ENZYMATIC COMPOSITION, PROCESSES TO DEGRAD A CELLULOSIC MATERIAL, TO SYNTHESIZE A FERMENTATION PRODUCT, AND, TO FERMENT A CLEAN MATERIAL
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US15/265,460 US10081824B2 (en) 2011-08-24 2016-09-14 Cellulolytic enzyme compositions and uses thereof
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Cited By (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013148504A2 (en) 2012-03-26 2013-10-03 Novozymes North America, Inc. Methods of preconditioning cellulosic material
WO2014058896A1 (en) 2012-10-08 2014-04-17 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014060378A1 (en) * 2012-10-16 2014-04-24 Dsm Ip Assets B.V. Cell wall deconstruction enzymes of pseudocercosporella herpotrichoides and|uses thereof
WO2014066141A2 (en) 2012-10-24 2014-05-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014082565A1 (en) 2012-11-27 2014-06-05 Novozymes A/S Milling process
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WO2014085439A1 (en) 2012-11-30 2014-06-05 Novozymes A/S Processes for producing fermentation products
WO2014092832A2 (en) 2012-09-19 2014-06-19 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2014093835A1 (en) 2012-12-14 2014-06-19 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014099798A1 (en) 2012-12-19 2014-06-26 Novozymes A/S Polypeptides having cellulolytic enhancinc activity and polynucleotides encoding same
WO2014138672A1 (en) 2013-03-08 2014-09-12 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2014182990A1 (en) 2013-05-10 2014-11-13 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2015035029A1 (en) 2013-09-04 2015-03-12 Novozymes A/S Processes for increasing enzymatic hydrolysis of cellulosic material
WO2015066492A1 (en) 2013-11-01 2015-05-07 Novozymes A/S Methods of saccharifying and fermenting a cellulosic material
WO2015081139A1 (en) 2013-11-26 2015-06-04 Novozymes A/S Enzyme compositions and uses thereof
WO2015105835A1 (en) 2014-01-07 2015-07-16 Novozymes A/S Process for degrading mannan-containing cellulosic materials
WO2015144782A1 (en) * 2014-03-25 2015-10-01 Novozymes A/S Dishwashing composition containing cellulytic enzymes and use thereof
WO2015187935A1 (en) 2014-06-06 2015-12-10 Novozymes A/S Enzyme compositions and uses thereof
WO2016029107A1 (en) 2014-08-21 2016-02-25 Novozymes A/S Process for saccharifying cellulosic material under oxygen addition
WO2016037096A1 (en) 2014-09-05 2016-03-10 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
CN105431528A (en) * 2013-08-30 2016-03-23 诺维信公司 Enzyme composition and uses thereof
WO2016045569A1 (en) 2014-09-23 2016-03-31 Novozymes A/S Processes for producing ethanol and fermenting organisms
WO2016138167A2 (en) 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016135351A1 (en) 2015-06-30 2016-09-01 Novozymes A/S Laundry detergent composition, method for washing and use of composition
WO2016145358A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Enzymatic hydrolysis with hemicellulolytic enzymes
WO2016145350A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass
WO2016145084A1 (en) 2015-03-09 2016-09-15 Novozymes A/S Methods of introducing multiple expression constructs into a eukaryotic cell
WO2016145363A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass employing an oxidoreductase with an aa9 polypeptide
WO2016188459A1 (en) 2015-05-27 2016-12-01 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2016205127A1 (en) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
WO2017019491A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2017019490A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having arabinofuranosidase activity and polynucleotides encoding same
WO2017040907A1 (en) 2015-09-04 2017-03-09 Novozymes A/S Methods of inhibiting aa9 lytic polysaccharide monooxygenase catalyzed inactivation of enzyme compositions
WO2017050242A1 (en) 2015-09-22 2017-03-30 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017070219A1 (en) 2015-10-20 2017-04-27 Novozymes A/S Lytic polysaccharide monooxygenase (lpmo) variants and polynucleotides encoding same
WO2017076421A1 (en) 2015-11-02 2017-05-11 Renescience A/S Solubilization of msw with blend enzymes
WO2017097895A1 (en) * 2015-12-10 2017-06-15 Dsm Ip Assets B.V. Beta-glucosidase and uses thereof
WO2017107980A1 (en) 2015-12-23 2017-06-29 Novozymes A/S Methods for enhancing the dewaterability of sludge with enzyme treatment
WO2017151957A1 (en) 2016-03-02 2017-09-08 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017165760A1 (en) 2016-03-24 2017-09-28 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017205535A1 (en) 2016-05-27 2017-11-30 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018026868A1 (en) 2016-08-01 2018-02-08 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018085370A1 (en) 2016-11-02 2018-05-11 Novozymes A/S Processes for reducing production of primeverose during enzymatic saccharification of lignocellulosic material
WO2018098381A1 (en) 2016-11-23 2018-05-31 Novozymes A/S Improved yeast for ethanol production
WO2018106792A1 (en) 2016-12-06 2018-06-14 Novozymes A/S Improved processes for production of ethanol from xylose-containing cellulosic substrates using engineered yeast strains
WO2018220116A1 (en) 2017-05-31 2018-12-06 Novozymes A/S Xylose fermenting yeast strains and processes thereof for ethanol production
WO2018222990A1 (en) 2017-06-02 2018-12-06 Novozymes A/S Improved yeast for ethanol production
WO2019005755A1 (en) 2017-06-28 2019-01-03 Novozymes A/S Polypeptides having trehalase activity and polynucleotides encoding same
WO2019030165A1 (en) 2017-08-08 2019-02-14 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
WO2019148192A1 (en) 2018-01-29 2019-08-01 Novozymes A/S Microorganisms with improved nitrogen utilization for ethanol production
WO2019161227A1 (en) 2018-02-15 2019-08-22 Novozymes A/S Improved yeast for ethanol production
WO2019201765A1 (en) 2018-04-20 2019-10-24 Renescience A/S Method for determining chemical compounds in waste
WO2019241615A1 (en) 2018-06-14 2019-12-19 Ecolab Usa Inc. Synergistic cellulase-surfactant interactions for degradation of bacterial cellulose
WO2019241614A1 (en) 2018-06-14 2019-12-19 Ecolab Usa Inc. Compositions comprising enzyme and quaternary ammonium compounds
WO2020023411A1 (en) 2018-07-25 2020-01-30 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020076697A1 (en) 2018-10-08 2020-04-16 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020123845A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Methods for increasing the productivity of a filamentous fungal cell in the production of a polypeptide
WO2020123463A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2020206058A1 (en) 2019-04-02 2020-10-08 Novozymes A/S Process for producing a fermentation product
WO2021021458A1 (en) 2019-07-26 2021-02-04 Novozymes A/S Microorganisms with improved nitrogen transport for ethanol production
WO2021025872A1 (en) 2019-08-06 2021-02-11 Novozymes A/S Fusion proteins for improved enzyme expression
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WO2022261003A1 (en) 2021-06-07 2022-12-15 Novozymes A/S Engineered microorganism for improved ethanol fermentation
WO2024064901A2 (en) 2022-09-23 2024-03-28 Novozymes A/S Improved fermenting organism for ethanol production
WO2024113151A1 (en) 2022-11-29 2024-06-06 Ecolab Usa Inc. A controlled release cleansing tablet containing biological actives
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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112014004193A2 (en) * 2011-08-24 2017-03-28 Novozymes Inc trichoderma recombinant host cell, methods for producing an enzyme composition, enzyme composition, processes for degrading a cellulosic material, for synthesizing a fermentation product, and for fermenting a cellulosic material
US11623885B2 (en) 2015-12-23 2023-04-11 Novozymes A/S Methods for enhancing the dewaterability of sludge with enzyme treatment
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
EP3874034A1 (en) * 2018-10-29 2021-09-08 Roberto Bassi Transgenic microalgae for the production of plant cell wall degrading enzymes having heat-stable cellulolytic activity

Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
WO1990015861A1 (en) 1989-06-13 1990-12-27 Genencor International, Inc. A method for killing cells without cell lysis
WO1991005039A1 (en) 1989-09-26 1991-04-18 Midwest Research Institute Thermostable purified endoglucanases from thermophilic bacterium acidothermus cellulolyticus
WO1993015186A1 (en) 1992-01-27 1993-08-05 Midwest Research Institute Thermostable purified endoglucanases from thermophilic bacterium acidothermus cellulolyticus
WO1994021785A1 (en) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes with xylanase activity from aspergillus aculeatus
WO1995033836A1 (en) 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Phosphonyldipeptides useful in the treatment of cardiovascular diseases
WO1996000787A1 (en) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic fusarium expression system and promoters and terminators for use therein
WO1996002551A1 (en) 1994-07-15 1996-02-01 Midwest Research Institute Gene coding for the e1 endoglucanase
WO1997026330A2 (en) 1996-01-19 1997-07-24 Novo Nordisk Biotech, Inc. Morphological mutants of filamentous fungi
US6011147A (en) 1986-04-30 2000-01-04 Rohm Enzyme Finland Oy Fungal promoters active in the presence of glucose
WO2000024883A1 (en) 1998-10-26 2000-05-04 Novozymes A/S Constructing and screening a dna library of interest in filamentous fungal cells
WO2000056900A2 (en) 1999-03-22 2000-09-28 Novo Nordisk Biotech, Inc. Promoter sequences derived from fusarium venenatum and uses thereof
WO2000070031A1 (en) 1999-05-19 2000-11-23 Midwest Research Institute E1 endoglucanase variants y245g, y82r and w42r
US20020164730A1 (en) 2000-02-24 2002-11-07 Centro De Investigaciones Energeticas, Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Procedure for the production of ethanol from lignocellulosic biomass using a new heat-tolerant yeast
WO2002095014A2 (en) 2001-05-18 2002-11-28 Novozymes A/S Polypeptides having cellobiase activity and polynucleotides encoding same
US6489127B1 (en) 2000-01-14 2002-12-03 Exelixis, Inc. Methods for identifying anti-cancer drug targets
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6511824B1 (en) 1999-03-17 2003-01-28 Exelixis, Inc. Nucleic acids and polypeptides of invertebrate TWIK channels and methods of use
US6515109B1 (en) 2000-10-12 2003-02-04 Exelixis, Inc. Human ECT2 polypeptide
WO2003062430A1 (en) 2002-01-23 2003-07-31 Royal Nedalco B.V. Fermentation of pentose sugars
US20040248258A1 (en) 2003-05-02 2004-12-09 Novozymes Biotech, Inc. Methods for producing secreted polypeptides
WO2005047499A1 (en) 2003-10-28 2005-05-26 Novozymes Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2005074647A2 (en) 2004-01-30 2005-08-18 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2005074656A2 (en) 2004-02-06 2005-08-18 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2005093050A2 (en) 2004-03-25 2005-10-06 Genencor International, Inc. Cellulase fusion protein and heterologous cellulase fusion construct encoding the same
WO2006032282A1 (en) 2004-09-24 2006-03-30 Cambi Bioethanol Aps Method for treating biomass and organic waste with the purpose of generating desired biologically based products
WO2006078256A2 (en) 2004-02-12 2006-07-27 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2006110901A2 (en) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain fermentable sugars
WO2008151043A1 (en) 2007-05-31 2008-12-11 Novozymes, Inc. Methods of increasing the cellulolytic enhancing activity of a polypeptide
WO2010039889A2 (en) 2008-09-30 2010-04-08 Novozymes, Inc. Methods for using positively and negatively selectable genes in a filamentous fungal cell
WO2010039840A1 (en) 2008-09-30 2010-04-08 Novozymes, Inc. Methods for producing polypeptides in enzyme-deficient mutants of fusarium venenatum
WO2010059424A2 (en) * 2008-11-18 2010-05-27 Novozymes, Inc. Methods and compositions for degrading cellulosic material
WO2010096673A1 (en) 2009-02-20 2010-08-26 Danisco Us Inc. Fermentation broth formulations
WO2011041397A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011057086A1 (en) * 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011057140A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2011075677A2 (en) 2009-12-18 2011-06-23 Novozymes, Inc. Methods for producing polypeptides in protease-deficient mutants of trichoderma

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2649188A1 (en) * 2010-12-06 2013-10-16 Novozymes North America, Inc. Methods of hydrolyzing oligomers in hemicellulosic liquor
MX337942B (en) * 2011-01-26 2016-03-29 Novozymes As Polypeptides having endoglucanase activity and polynucleotides encoding same.

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
US6011147A (en) 1986-04-30 2000-01-04 Rohm Enzyme Finland Oy Fungal promoters active in the presence of glucose
WO1990015861A1 (en) 1989-06-13 1990-12-27 Genencor International, Inc. A method for killing cells without cell lysis
WO1991005039A1 (en) 1989-09-26 1991-04-18 Midwest Research Institute Thermostable purified endoglucanases from thermophilic bacterium acidothermus cellulolyticus
US5275944A (en) 1989-09-26 1994-01-04 Midwest Research Institute Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068
US5536655A (en) 1989-09-26 1996-07-16 Midwest Research Institute Gene coding for the E1 endoglucanase
WO1993015186A1 (en) 1992-01-27 1993-08-05 Midwest Research Institute Thermostable purified endoglucanases from thermophilic bacterium acidothermus cellulolyticus
WO1994021785A1 (en) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes with xylanase activity from aspergillus aculeatus
WO1995033836A1 (en) 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Phosphonyldipeptides useful in the treatment of cardiovascular diseases
WO1996000787A1 (en) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic fusarium expression system and promoters and terminators for use therein
WO1996002551A1 (en) 1994-07-15 1996-02-01 Midwest Research Institute Gene coding for the e1 endoglucanase
WO1997026330A2 (en) 1996-01-19 1997-07-24 Novo Nordisk Biotech, Inc. Morphological mutants of filamentous fungi
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
WO2000024883A1 (en) 1998-10-26 2000-05-04 Novozymes A/S Constructing and screening a dna library of interest in filamentous fungal cells
US6511824B1 (en) 1999-03-17 2003-01-28 Exelixis, Inc. Nucleic acids and polypeptides of invertebrate TWIK channels and methods of use
WO2000056900A2 (en) 1999-03-22 2000-09-28 Novo Nordisk Biotech, Inc. Promoter sequences derived from fusarium venenatum and uses thereof
WO2000070031A1 (en) 1999-05-19 2000-11-23 Midwest Research Institute E1 endoglucanase variants y245g, y82r and w42r
US6489127B1 (en) 2000-01-14 2002-12-03 Exelixis, Inc. Methods for identifying anti-cancer drug targets
US20020164730A1 (en) 2000-02-24 2002-11-07 Centro De Investigaciones Energeticas, Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Procedure for the production of ethanol from lignocellulosic biomass using a new heat-tolerant yeast
US6515109B1 (en) 2000-10-12 2003-02-04 Exelixis, Inc. Human ECT2 polypeptide
WO2002095014A2 (en) 2001-05-18 2002-11-28 Novozymes A/S Polypeptides having cellobiase activity and polynucleotides encoding same
WO2003062430A1 (en) 2002-01-23 2003-07-31 Royal Nedalco B.V. Fermentation of pentose sugars
US20040248258A1 (en) 2003-05-02 2004-12-09 Novozymes Biotech, Inc. Methods for producing secreted polypeptides
WO2005047499A1 (en) 2003-10-28 2005-05-26 Novozymes Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2005074647A2 (en) 2004-01-30 2005-08-18 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2005074656A2 (en) 2004-02-06 2005-08-18 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2006078256A2 (en) 2004-02-12 2006-07-27 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2005093050A2 (en) 2004-03-25 2005-10-06 Genencor International, Inc. Cellulase fusion protein and heterologous cellulase fusion construct encoding the same
WO2006032282A1 (en) 2004-09-24 2006-03-30 Cambi Bioethanol Aps Method for treating biomass and organic waste with the purpose of generating desired biologically based products
WO2006110900A2 (en) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain ethanol
WO2006110901A2 (en) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain fermentable sugars
WO2006110899A2 (en) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Integration of alternative feedstreams in biomass treatment and utilization
WO2006110891A2 (en) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain a target chemical
WO2008151043A1 (en) 2007-05-31 2008-12-11 Novozymes, Inc. Methods of increasing the cellulolytic enhancing activity of a polypeptide
WO2010039889A2 (en) 2008-09-30 2010-04-08 Novozymes, Inc. Methods for using positively and negatively selectable genes in a filamentous fungal cell
WO2010039840A1 (en) 2008-09-30 2010-04-08 Novozymes, Inc. Methods for producing polypeptides in enzyme-deficient mutants of fusarium venenatum
WO2010059424A2 (en) * 2008-11-18 2010-05-27 Novozymes, Inc. Methods and compositions for degrading cellulosic material
WO2010096673A1 (en) 2009-02-20 2010-08-26 Danisco Us Inc. Fermentation broth formulations
WO2011041397A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011057086A1 (en) * 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011057140A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2011075677A2 (en) 2009-12-18 2011-06-23 Novozymes, Inc. Methods for producing polypeptides in protease-deficient mutants of trichoderma

Non-Patent Citations (124)

* Cited by examiner, † Cited by third party
Title
AIKAWA ET AL., J. BIOCHEM., vol. 129, 2001, pages 791 - 794
ALFENORE ET AL.: "Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch. process", 2002, SPRINGER-VERLAG
ALIZADEH ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 121, 2005, pages 1133 - 1141
BAILEY; BIELY; POUTANEN: "Interlaboratory testing of methods for assay of xylanase activity", JOURNAL OF BIOTECHNOLOGY, vol. 23, no. 3, 1992, pages 257 - 270, XP023704921, DOI: doi:10.1016/0168-1656(92)90074-J
BALLESTEROS ET AL., BIOCHEM. BIOTECHNOL., vol. 129-132, 2006, pages 496 - 508
BEALL ET AL.: "Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli", BIOTECH. BIOENG., vol. 38, 1991, pages 296 - 303, XP000215006, DOI: doi:10.1002/bit.260380311
BECKER; GUARENTE: "Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology", vol. 194, ACADEMIC PRESS, INC., pages: 182 - 187
BIELY; PUCHARD: "Recent progress in the assays of xylanolytic enzymes", JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, vol. 86, no. 11, 2006, pages 1636 - 1647
CHANDRA ET AL.: "Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?", ADV. BIOCHEM. ENGIN./BIOTECHNOL., vol. 108, 2007, pages 67 - 93
CHEN, R.; LEE, Y. Y.: "Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass", APPL. BIOCHEM. BIOTECHNOL., vol. 63-65, 1997, pages 435 - 448, XP008087560, DOI: doi:10.1007/BF02920444
CHEN; HO: "Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae", APPL. BIOCHEM. BIOTECHNOL., vol. 39-40, 1993, pages 135 - 147, XP009063851, DOI: doi:10.1007/BF02918984
CHRISTENSEN ET AL., BIOFTECHNOLOGY, vol. 6, 1988, pages 1419 - 1422
CHRISTENSEN ET AL., BIOLTECHNOLOGY, vol. 6, 1988, pages 1419 - 1422
CHUNDAWAT ET AL., BIOTECHNOL. BIOENG., vol. 96, 2007, pages 219 - 231
CULLEN ET AL., NUCLEIC ACIDS RES., vol. 15, 1987, pages 9163 - 9175
DE VRIES, J. BACTERIOL., vol. 180, 1998, pages 243 - 249
DEANDA ET AL.: "Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering", APPL. ENVIRON. MICROBIOL., vol. 62, 1996, pages 4465 - 4470
DIENES ET AL., ENZYME AND MICROBIAL TECHNOLOGY, vol. 40, 2007, pages 1087 - 1094
DUFF; MURRAY, BIORESOURCE TECHNOLOGY, vol. 855, 1996, pages 1 - 33
EBRINGEROVA ET AL., ADV. POLYM. SCI., vol. 186, 2005, pages 1 - 67
EDGAR, NUCLEIC ACIDS RESEARCH, vol. 32, 2004, pages 1792 - 1797
EZEJI, T. C.; QURESHI, N.; BLASCHEK, H. P.: "Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 19, no. 6, 2003, pages 595 - 603
FERNANDA DE CASTILHOS CORAZZA; FIAVIO FARIA DE MORAES; GISELLA MARIA ZANIN; IVO NEITZEL: "Optimal control in fed-batch reactor for the cellobiose hydrolysis", ACTA SCIENTIARUM. TECHNOLOGY, vol. 25, 2003, pages 33 - 38
GALBE; ZACCHI, APPL. MICROBIOL. BIOTECHNOL., vol. 59, 2002, pages 618 - 628
GALBE; ZACCHI: "Pretreatment of lignocellulosic materials for efficient bioethanol production", ADV. BIOCHEM. ENGIN.LBIOTECHNOL., vol. 108, 2007, pages 41 - 65, XP008113337, DOI: doi:10.1007/10_2007_070
GAO D ET AL: "Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover", BIORESOURCE TECHNOLOGY, ELSEVIER BV, GB, vol. 101, no. 8, 1 April 2010 (2010-04-01), pages 2770 - 2781, XP026833855, ISSN: 0960-8524, [retrieved on 20091130] *
GEMS ET AL., GENE, vol. 98, 1991, pages 61 - 67
GHOSE, PURE AND APPL. CHEM., vol. 59, 1987, pages 257 - 268
GHOSE: "Measurement of cellulase activities", PURE APPL. CHEM., vol. 59, 1987, pages 257 - 68, XP000652082
GHOSE; BISARIA, PURE & APPL. CHEM., vol. 59, 1987, pages 1739 - 1752
GHOSH; SINGH: "Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass", ADV. APPL. MICROBIOL., vol. 39, 1993, pages 295 - 333, XP009102696, DOI: doi:10.1016/S0065-2164(08)70598-7
GIESECKE ET AL., JOURNAL OF VIROLOGY METHODS, vol. 38, 1992, pages 47 - 60
GOLLAPALLI ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 98, 2002, pages 23 - 35
GONG, C. S.; CAO, N. J.; DU, J.; TSAO, G. T.: "Advances in Biochemical EngineeringBiotechno1ogv", vol. 65, 1999, SPRINGER-VERLAG, article "Ethanol production from renewable resources", pages: 207 - 241
GONG, C. S.; CAO, N. J.; DU, J.; TSAO, G. T.: "Advances in Biochemical EngineeringlBiotechnology", vol. 65, 1999, SPRINGER-VERLAG, article "Ethanol production from renewable resources", pages: 207 - 241
GOUTAMI BANERJEE ET AL: "Synthetic enzyme mixtures for biomass deconstruction: Production and optimization of a core set", BIOTECHNOLOGY AND BIOENGINEERING, WILEY & SONS, HOBOKEN, NJ, US, vol. 106, no. 5, 1 August 2010 (2010-08-01), pages 707 - 720, XP002660862, ISSN: 0006-3592, [retrieved on 20100326], DOI: 10.1002/BIT.22741 *
GUNASEELAN V.N., BIOMASS AND BIOENERGY, vol. 13, no. 1-2, 1997, pages 83 - 114
GUSAKOV, A. V.; SINITSYN, A. P.: "Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process", ENZ. MICROB. TECHNOL., vol. 7, 1985, pages 346 - 352, XP023679176, DOI: doi:10.1016/0141-0229(85)90114-0
GUSAKOV, A. V.; SINITSYN, A. P.; DAVYDKIN, . Y.; DAVYDKIN, V. Y.; PROTAS, O. V.: "Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field", APPL. BIOCHEM. BIOTECHNOL., vol. 56, 1996, pages 141 - 153, XP035176121, DOI: doi:10.1007/BF02786945
HENDRIKS; ZEEMAN: "Pretreatments to enhance the digestibility of lignocellulosic biomass", BIORESOURCE TECHNOL., vol. 100, 2009, pages 10 - 18, XP025407559, DOI: doi:10.1016/j.biortech.2008.05.027
HENRISSAT B.: "A classification of glycosyl hydrolases based on amino-acid sequence similarities", BIOCHEM. J., vol. 280, 1991, pages 309 - 316
HENRISSAT B.; BAIROCH A.: "Updating the sequence-based classification of glycosyl hydrolases", BIOCHEM. J., vol. 316, 1996, pages 695 - 696, XP001176681
HERRMANN; VRSANSKA; JURICKOVA; HIRSCH; BIELY; KUBICEK: "The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase", BIOCHEMICAL JOUMAL, vol. 321, 1997, pages 375 - 381
HINNEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 75, 1978, pages 1920
HO ET AL.: "Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose", APPL. ENVIRON. MICROBIOL., vol. 64, 1998, pages 1852 - 1859
HSU, T.-A.: "Handbook on Bioethanol: Production and Utilization", 1996, TAYLOR & FRANCIS, article "Pretreatment of biomass", pages: 179 - 212
INGRAM ET AL.: "Metabolic engineering of bacteria for ethanol production", BIOTECHNOL. BIOENG., vol. 58, 1998, pages 204 - 214, XP000999111, DOI: doi:10.1002/(SICI)1097-0290(19980420)58:2/3<204::AID-BIT13>3.0.CO;2-C
INNIS ET AL.: "PCR: A Guide to Methods and Application", 1990, ACADEMIC PRESS
ITO ET AL., J. BACTERIOL., vol. 153, 1983, pages 163
JANSON AND RYDEN: "Protein Purification", 1989, VCH PUBLISHERS
K. JACQUES, T.P. LYONS AND D.R. KELSALL: "The Alcohol Textbook", 1999, NOTTINGHAM UNIVERSITY PRESS
KATAOKA, N.; A. MIYA; K. KIRIYAMA: "Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria", WATER SCIENCE AND TECHNOLOGY, vol. 36, no. 6-7, 1997, pages 41 - 47
KATOH ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 537, 2009, pages 39 - 64
KATOH ET AL., NUCLEIC ACIDS RESEARCH, vol. 33, 2005, pages 511 - 518
KATOH; KUMA, NUCLEIC ACIDS RESEARCH, vol. 30, 2002, pages 3059 - 3066
KATOH; TOH, BIOINFORMATICS, vol. 23, 2007, pages 372 - 374
KATOH; TOH, BIOINFORMATICS, vol. 26, 2010, pages 1899 - 1900
KOTTER; CIRIACY: "Xylose fermentation by Saccharomyces cerevisiae", APPL. MICROBIOL. BIOTECHNOL., vol. 38, 1993, pages 776 - 783
KURABI ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 121, 2005, pages 219 - 230
KUYPER ET AL.: "Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle", FEMS YEAST RESEARCH, vol. 4, 2004, pages 655 - 664
LEE ET AL., ADV. BIOCHEM. ENG. BIOTECHNOL, vol. 65, 1999, pages 93 - 115
LEVER ET AL., ANAL. BIOCHEM., vol. 47, 1972, pages 273 - 279
LEVER: "A new reaction for colorimetric determination of carbohydrates", ANAL. BIOCHEM, vol. 47, 1972, pages 273 - 279, XP024820395, DOI: doi:10.1016/0003-2697(72)90301-6
LIN ET AL., APPL MICROBIOL. BIOTECHNOL., vol. 69, 2006, pages 627 - 642
LYND, APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 24/25, 1990, pages 695 - 719
LYND, L. R.; WEIMER, P. J.; VAN ZYL, W. H.; PRETORIUS, . S.: "Microbial cellulose utilization: Fundamentals and biotechnology", MICROBIOL. MOL. BIOL. REVIEWS, vol. 66, 2002, pages 506 - 577, XP002551605, DOI: doi:10.1128/MMBR.66.3.506-577.2002
MALARDIER ET AL., GENE, vol. 78, 1989, pages 147 - 156
MARTIN ET AL., J. CHEM. TECHNOL. BIOTECHNOL., vol. 81, 2006, pages 1669 - 1677
MCMILLAN, J. D.: "Enzymatic Conversion of Biomass for Fuels Production", 1994, ACS SYMPOSIUM SERIES 566, AMERICAN CHEMICAL SOCIETY, article "Pretreating lignocellulosic biomass: a review"
MONTENECOURT; EVELEIGH, ADV. CHEM. SER., vol. 181, 1979, pages 289 - 301
MOSIER ET AL., BIORESOURCE TECHNOL., vol. 96, 2005, pages 673 - 686
MOSIER ET AL., BIORESOURCE TECHNOLOGY, vol. 96, 2005, pages 673 - 686
MOSIER ET AL.: "Advances in Biochemical EngineeringlBiotechnology", vol. 65, 1999, article "Recent Progress in Bioconversion of Lignocellulosics", pages: 23 - 40
MOSIER ET AL.: "Features of promising technologies for pretreatment of lignocellulosic biomass", BIORESOURCE TECHNOL., vol. 96, 2005, pages 673 - 686, XP025313257, DOI: doi:10.1016/j.biortech.2004.06.025
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NIELSEN ET AL., PROTEIN ENGINEERING, vol. 10, 1997, pages 1 - 6
NIGAM, P.; SINGH, D.: "Processes for fermentative production of xylitol - a sugar substitute", PROCESS BIOCHEMISTRY, vol. 30, no. 2, 1995, pages 117 - 124
OKADA ET AL., APPL. ENVIRON. MICROBIOL., vol. 64, 1988, pages 555 - 563
OLSSON; HAHN-HAGERDAL: "Fermentation of lignocellulosic hydrolysates for ethanol production", ENZ. MICROB. TECH., vol. 18, 1996, pages 312 - 331, XP002312595, DOI: doi:10.1016/0141-0229(95)00157-3
OOI ET AL., NUCLEIC ACIDS RESEARCH, vol. 18, 1990, pages 5884
PALONEN ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 117, 2004, pages 1 - 17
PAN ET AL., BIOTECHNOL. BIOENG., vol. 90, 2005, pages 473 - 481
PAN ET AL., BIOTECHNOL. BIOENG., vol. 94, 2006, pages 851 - 861
PENTTILA ET AL., GENE, vol. 45, 1986, pages 253 - 263
PENTTILA ET AL., GENE, vol. 61, 1987, pages 155 - 164
PHILIPPIDIS, G. P.: "Handbook on Bioethanol: Production and Utilization", 1996, TAYLOR & FRANCIS, article "Cellulose bioconversion technology", pages: 179 - 212
RICE ET AL.: "EMBOSS: The European Molecular Biology Open Software Suite", TRENDS GENET., vol. 16, 2000, pages 276 - 277, XP004200114, DOI: doi:10.1016/S0168-9525(00)02024-2
RICHARD, A.; MARGARITIS, A.: "Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers", BIOTECHNOLOGY AND BIOENGINEERING, vol. 87, no. 4, 2004, pages 501 - 515
RYU, S. K.; LEE, J. M.: "Bioconversion of waste cellulose by using an attrition bioreactor", BIOTECHNOL BIOENG., vol. 25, 1983, pages 53 - 65, XP002397253, DOI: doi:10.1002/bit.260250106
SAARILAHTI ET AL., GENE, vol. 90, 1990, pages 9 - 14
SAKAMOTO ET AL., CURRENT GENETICS, vol. 27, 1995, pages 435 - 439
SALOHEIMO ET AL., GENE, vol. 63, 1988, pages 11 - 22
SALOHEIMO ET AL., MOLECULAR MICROBIOLOGY, vol. 13, 1994, pages 219 - 228
SASSNER ET AL., ENZYME MICROB. TECHNOL., vol. 39, 2006, pages 756 - 762
SCHELL ET AL., APPL. BIOCHEM. AND BIOTECHNOL., vol. 105, no. 108, 2003, pages 69 - 85
SCHELL ET AL., BIORESOURCE TECHNOL., vol. 91, 2004, pages 179 - 188
SCHMIDT; THOMSEN, BIORESOURCE TECHNOL., vol. 64, 1998, pages 139 - 151
SHALLOM, D.; SHOHAM, Y.: "Microbial hemicellulases", CURRENT OPINION IN MICROBIOLOGY, vol. 6, no. 3, 2003, pages 219 - 228, XP002666720, DOI: doi:10.1016/S1369-5274(03)00056-0
SHEEHAN, J.; HIMMEL, M.: "Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol", BIOTECHNOL. PROG., vol. 15, 1999, pages 817 - 827
SIEZEN; LEUNISSEN, PROTEIN SCIENCE, vol. 6, 1997, pages 501 - 523
SILVEIRA, M. M.; JONAS, R.: "The biotechnological production of sorbitol", APPL. MICROBIOL. BIOTECHNOL., vol. 59, 2002, pages 400 - 408
SPANIKOVA; BIELY: "Glucuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune", FEBS LETTERS, vol. 580, no. 19, 2006, pages 4597 - 4601, XP028061315, DOI: doi:10.1016/j.febslet.2006.07.033
SZECSI, SCAND. J. CLIN. LAB. IN VEST., vol. 210, 1992, pages 5 - 22
TAHERZADEH; KARIMI: "Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review", INT. J. OF MOL. SCI., vol. 9, 2008, pages 1621 - 1651, XP002674403, DOI: doi:10.3390/IJMS9091621
TEERI ET AL.: "Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?", BIOCHEM. SOC. TRANS., vol. 26, 1998, pages 173 - 178
TEERI: "Crystalline cellulose degradation: New insight into the function of cellobiohydrolases", TRENDS IN BIOTECHNOLOGY, vol. 15, 1997, pages 160 - 167, XP004059844, DOI: doi:10.1016/S0167-7799(97)01032-9
TEYMOURI ET AL., BIORESOURCE TECHNOL., vol. 96, 2005, pages 2014 - 2018
THOMPSON, NUCLEIC ACIDS RESEARCH, vol. 22, 1994, pages 4673 - 4680
TOMME ET AL., EUR. J. BIOCHEM., vol. 170, 1988, pages 575 - 581
VALLANDER; ERIKSSON: "Production of ethanol from lignocellulosic materials: State of the art", ADV. BIOCHEM. ENG.LBIOTECHNOL., vol. 42, 1990, pages 63 - 95
VAN TILBEURGH ET AL., FEBS LETTERS, vol. 149, 1982, pages 152 - 156
VAN TILBEURGH; CLAEYSSENS, FEBS LETTERS, vol. 187, 1985, pages 283 - 288
VARGA ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 113-116, 2004, pages 509 - 523
VARGA ET AL., BIOTECHNOL. BIOENG., vol. 88, 2004, pages 567 - 574
VENTURI ET AL.: "Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties", J. BASIC MICROBIOL., vol. 42, 2002, pages 55 - 66
WALFRIDSSON ET AL.: "Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase", APPL. ENVIRON. MICROBIOL., vol. 61, 1995, pages 4184 - 4190, XP002053306
WISELOGEL ET AL.: "Handbook on Bioethanol", 1995, TAYLOR & FRANCIS, pages: 105 - 118
WYMAN ET AL., BIORESOURCE TECHNOL., vol. 96, 2005, pages 1959 - 1966
WYMAN, BIORESOURCE TECHNOLOGY, vol. 50, 1994, pages 3 - 16
YANG; WYMAN: "Pretreatment: the key to unlocking low-cost cellulosic ethanol", BIOFUELS BIOPRODUCTS AND BIOREFINING-BIOFPR, vol. 2, 2008, pages 26 - 40, XP008150365, DOI: doi:10.1002/bbb.49
YELTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 1470 - 1474
ZHANG ET AL., BIOTECHNOLOGY ADVANCES, vol. 24, 2006, pages 452 - 481
ZHANG ET AL.: "Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis", SCIENCE, vol. 267, 1995, pages 240 - 243, XP002912123, DOI: doi:10.1126/science.267.5195.240
ZHANG ET AL.: "Outlook for cellulase improvement: Screening and selection strategies", BIOTECHNOLOGY ADVANCES, vol. 24, 2006, pages 452 - 481, XP028005978, DOI: doi:10.1016/j.biotechadv.2006.03.003

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2014092832A2 (en) 2012-09-19 2014-06-19 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2014058896A1 (en) 2012-10-08 2014-04-17 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3586610A1 (en) 2012-10-08 2020-01-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
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WO2014082565A1 (en) 2012-11-27 2014-06-05 Novozymes A/S Milling process
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WO2015105835A1 (en) 2014-01-07 2015-07-16 Novozymes A/S Process for degrading mannan-containing cellulosic materials
WO2015144782A1 (en) * 2014-03-25 2015-10-01 Novozymes A/S Dishwashing composition containing cellulytic enzymes and use thereof
WO2015187935A1 (en) 2014-06-06 2015-12-10 Novozymes A/S Enzyme compositions and uses thereof
WO2016029107A1 (en) 2014-08-21 2016-02-25 Novozymes A/S Process for saccharifying cellulosic material under oxygen addition
EP3805382A1 (en) 2014-08-28 2021-04-14 Renescience A/S Solubilization of msw with blend enzymes
EP3594335A1 (en) 2014-09-05 2020-01-15 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
WO2016037096A1 (en) 2014-09-05 2016-03-10 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
EP4406964A2 (en) 2014-09-05 2024-07-31 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
WO2016045569A1 (en) 2014-09-23 2016-03-31 Novozymes A/S Processes for producing ethanol and fermenting organisms
EP3739045A2 (en) 2015-02-24 2020-11-18 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016138167A2 (en) 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
EP4458964A2 (en) 2015-02-24 2024-11-06 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016145084A1 (en) 2015-03-09 2016-09-15 Novozymes A/S Methods of introducing multiple expression constructs into a eukaryotic cell
WO2016145350A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass
WO2016145363A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass employing an oxidoreductase with an aa9 polypeptide
WO2016145358A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Enzymatic hydrolysis with hemicellulolytic enzymes
WO2016188459A1 (en) 2015-05-27 2016-12-01 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2016205127A1 (en) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
WO2016135351A1 (en) 2015-06-30 2016-09-01 Novozymes A/S Laundry detergent composition, method for washing and use of composition
CN107922896A (en) * 2015-06-30 2018-04-17 诺维信公司 Laundry detergent composition, for washing method and composition purposes
WO2017019491A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2017019490A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having arabinofuranosidase activity and polynucleotides encoding same
WO2017040907A1 (en) 2015-09-04 2017-03-09 Novozymes A/S Methods of inhibiting aa9 lytic polysaccharide monooxygenase catalyzed inactivation of enzyme compositions
WO2017050242A1 (en) 2015-09-22 2017-03-30 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017070219A1 (en) 2015-10-20 2017-04-27 Novozymes A/S Lytic polysaccharide monooxygenase (lpmo) variants and polynucleotides encoding same
WO2017076421A1 (en) 2015-11-02 2017-05-11 Renescience A/S Solubilization of msw with blend enzymes
US10808235B2 (en) 2015-12-10 2020-10-20 Dsm Ip Assets B.V. Beta-glucosidase and uses thereof
WO2017097895A1 (en) * 2015-12-10 2017-06-15 Dsm Ip Assets B.V. Beta-glucosidase and uses thereof
EP3702469A1 (en) * 2015-12-10 2020-09-02 DSM IP Assets B.V. Beta-glucosidase and uses thereof
WO2017107980A1 (en) 2015-12-23 2017-06-29 Novozymes A/S Methods for enhancing the dewaterability of sludge with enzyme treatment
EP4410974A2 (en) 2016-03-02 2024-08-07 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017151957A1 (en) 2016-03-02 2017-09-08 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017165760A1 (en) 2016-03-24 2017-09-28 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017205535A1 (en) 2016-05-27 2017-11-30 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018026868A1 (en) 2016-08-01 2018-02-08 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018085370A1 (en) 2016-11-02 2018-05-11 Novozymes A/S Processes for reducing production of primeverose during enzymatic saccharification of lignocellulosic material
WO2018098381A1 (en) 2016-11-23 2018-05-31 Novozymes A/S Improved yeast for ethanol production
WO2018106792A1 (en) 2016-12-06 2018-06-14 Novozymes A/S Improved processes for production of ethanol from xylose-containing cellulosic substrates using engineered yeast strains
WO2018220116A1 (en) 2017-05-31 2018-12-06 Novozymes A/S Xylose fermenting yeast strains and processes thereof for ethanol production
WO2018222990A1 (en) 2017-06-02 2018-12-06 Novozymes A/S Improved yeast for ethanol production
EP4015524A1 (en) 2017-06-28 2022-06-22 Novozymes A/S Polypeptides having trehalase activity and polynucleotides encoding same
WO2019005755A1 (en) 2017-06-28 2019-01-03 Novozymes A/S Polypeptides having trehalase activity and polynucleotides encoding same
WO2019030165A1 (en) 2017-08-08 2019-02-14 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
WO2019148192A1 (en) 2018-01-29 2019-08-01 Novozymes A/S Microorganisms with improved nitrogen utilization for ethanol production
WO2019161227A1 (en) 2018-02-15 2019-08-22 Novozymes A/S Improved yeast for ethanol production
WO2019201765A1 (en) 2018-04-20 2019-10-24 Renescience A/S Method for determining chemical compounds in waste
WO2019241615A1 (en) 2018-06-14 2019-12-19 Ecolab Usa Inc. Synergistic cellulase-surfactant interactions for degradation of bacterial cellulose
WO2019241614A1 (en) 2018-06-14 2019-12-19 Ecolab Usa Inc. Compositions comprising enzyme and quaternary ammonium compounds
WO2020023411A1 (en) 2018-07-25 2020-01-30 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020076697A1 (en) 2018-10-08 2020-04-16 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020123463A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2020123845A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Methods for increasing the productivity of a filamentous fungal cell in the production of a polypeptide
WO2020206058A1 (en) 2019-04-02 2020-10-08 Novozymes A/S Process for producing a fermentation product
WO2021021458A1 (en) 2019-07-26 2021-02-04 Novozymes A/S Microorganisms with improved nitrogen transport for ethanol production
WO2021025872A1 (en) 2019-08-06 2021-02-11 Novozymes A/S Fusion proteins for improved enzyme expression
WO2021048164A1 (en) 2019-09-10 2021-03-18 Dsm Ip Assets B.V. Enzyme composition
WO2021055395A1 (en) 2019-09-16 2021-03-25 Novozymes A/S Polypeptides having beta-glucanase activity and polynucleotides encoding same
WO2021119304A1 (en) 2019-12-10 2021-06-17 Novozymes A/S Microorganism for improved pentose fermentation
WO2021148278A1 (en) 2020-01-24 2021-07-29 Novozymes A/S Mutants of a filamentous fungal cell having increased productivity in the production of a polypeptide
WO2021163030A2 (en) 2020-02-10 2021-08-19 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
WO2022049250A1 (en) 2020-09-04 2022-03-10 Novozymes A/S Improved fermenting organism for ethanol production
WO2022090564A1 (en) 2020-11-02 2022-05-05 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
WO2022096406A1 (en) 2020-11-04 2022-05-12 Renescience A/S Method for enzymatic and/or microbial processing of waste comprising recirculation of process water
WO2022096517A1 (en) 2020-11-04 2022-05-12 Renescience A/S Method for sanitizing waste
WO2022214457A1 (en) 2021-04-06 2022-10-13 Dsm Ip Assets B.V. Enzyme composition
WO2022214458A1 (en) 2021-04-06 2022-10-13 Dsm Ip Assets B.V. Enzyme composition
WO2022214459A1 (en) 2021-04-06 2022-10-13 Dsm Ip Assets B.V. Enzyme composition
WO2022214460A1 (en) 2021-04-08 2022-10-13 Dsm Ip Assets B.V. Process for the preparation of a sugar product and a fermentation product
WO2022261003A1 (en) 2021-06-07 2022-12-15 Novozymes A/S Engineered microorganism for improved ethanol fermentation
WO2024064901A2 (en) 2022-09-23 2024-03-28 Novozymes A/S Improved fermenting organism for ethanol production
WO2024113151A1 (en) 2022-11-29 2024-06-06 Ecolab Usa Inc. A controlled release cleansing tablet containing biological actives
WO2024115642A1 (en) 2022-12-01 2024-06-06 Renescience A/S Method for production of hydrogen gas from waste

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