CN118302534A

CN118302534A - Reduction of residual DNA in microbial fermentation products

Info

Publication number: CN118302534A
Application number: CN202280078274.5A
Authority: CN
Inventors: C·约尔特; R·库卡列夫斯基; J·斯文特; C·L·克里斯滕森; K·B·雅各布森
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2021-12-23
Filing date: 2022-12-23
Publication date: 2024-07-05

Abstract

The present invention provides a method for reducing the amount of DNA in a microbial fermentation product by adding a fungal dnase from aspergillus oryzae to the resulting fermentation product during the recovery process.

Description

Reduction of residual DNA in microbial fermentation products

Reference to sequence Listing

The application comprises a sequence listing in computer readable form. The computer readable form is incorporated herein by reference.

Technical Field

The present invention relates to the use of fungal dnase to reduce residual recombinant DNA in microbial fermentation products.

Background

The production of protein products by fermentation is a well known process and it is used to produce a variety of different proteins of interest on an industrial scale. During fermentation, some host cells producing the protein product of interest will rupture and the cellular content, including DNA, will be released into the fermentation broth. Furthermore, in some fermentations, the protein of interest is produced as an intracellular product. This means that the cells must be destroyed/lysed, for example by homogenization, before the recovery and purification process after fermentation, and this inevitably leads to a significant amount of DNA being released into the fermentation broth, which can then finally be taken as residual DNA into the final protein product.

For example, for environmental or health concerns, it may be desirable to avoid residual DNA from host cells producing the protein of interest. This is a problem where recombinant DNA requires special attention.

Thus, there is a need to reduce residual DNA in the fermentation product.

Disclosure of Invention

In a first aspect, the present invention provides a method for reducing the amount of DNA in a microbial fermentation product, the method comprising

(A) Providing a fermentation broth comprising microbial host cells, recombinant DNA from these microbial host cells, and a protein of interest produced by these microbial host cells;

(b) Subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; and

(C) Subjecting the fermentation broth supernatant to a membrane filtration step to provide a fermentation product, wherein the membrane has a size exclusion limit of less than 100kDa or less than 1 μm;

Wherein the fungal dnase or variant thereof is added to the fermentation broth before, during or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c).

In another aspect, the invention provides a microbial fermentation product comprising less than 10ng/g, less than 5ng/g, less than 1ng/g, or less than 0.1ng/g recombinant DNA.

Other aspects and embodiments of the invention will be apparent from the specification and examples.

Unless otherwise indicated, or other meanings apparent from the context, all percentages are by weight (% w/w).

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Definition of the definition

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For the purposes of the present invention, the sequence identity between two amino acid sequences is determined as output of the "longest identity" using the Needman-Wen application algorithm (Needleman-Wunsch algorithm) (Needleman and Wunsch,1970, J.mol. Biol. [ J.Mole. Mol. Biol. ] 48:443-453) as implemented in the Nidel (Needle) program of the EMBOSS software package (EMBOSS: the European Molecular Biology Open Software Suite [ European molecular biology open software suite ], rice et al 2000,Trends Genet. [ genetics trend ] 16:276-277), preferably version 6.6.0 or newer. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. In order for the Needle program to report the longest identity, the-nobrief option must be specified in the command line. The output of the "longest identity" of the Needle label is calculated as follows:

(identical residue. Times.100)/(alignment Length-total number of gaps in the alignment)

Cell debris: the term "cell debris" refers to cell walls and other insoluble cellular components that are released after cell wall/membrane disruption, e.g., after microbial cell disruption/lysis/homogenization. Even without deliberate disruption of the cell wall/membrane, some microbial cells may rupture and release cell debris during fermentation.

Sequence(s)

SEQ ID NO. 1: amino acid sequence of dnase from bacillus food (Bacillus cibi).

SEQ ID NO. 2: amino acid sequence of dnase from aspergillus oryzae (Aspergillus oryzae)

SEQ ID NO. 3: amino acid sequence of NUC3 dnase motif.

SEQ ID NO. 4: amino acid sequence of NUC3 dnase motif.

SEQ ID NO. 5-15: nucleotide sequences of primers and probes for dPCR (example 1).

Detailed Description

We have found that fungal DNases, particularly NUC3 nucleases, are highly effective in reducing or removing residual DNA in fermentation products.

The present invention provides methods for reducing the amount of residual DNA in products comprising a protein of interest produced by fermentation of a microbial host cell (particularly a recombinant host cell) that expresses the protein of interest and secretes it into the fermentation broth or accumulates it as an intracellular product. If the protein of interest is produced as an intracellular product, the microbial host cell may be homogenized prior to recovery and purification.

After fermentation and optional homogenization, the fermentation broth is subjected to a flocculation or precipitation step to provide a fermentation broth supernatant. This is an efficient solid/liquid separation that removes most (insoluble) host cells and cell debris and retains the protein of interest and some aqueous solution of soluble host cell components (e.g., residual host cell DNA).

Finally, the fermentation broth supernatant is filtered during membrane filtration to increase the purity of the protein of interest and provide a liquid fermentation product. Membrane filtration may remove higher and/or lower molecular weight components depending on the type of membrane and filtration process. Some membrane filtration processes retain filtrate (microfiltration) while others retain retentate/permeate (ultrafiltration).

The methods of the invention include adding a fungal dnase or variant thereof to the fermentation medium prior to or during fermentation, to the fermentation broth prior to flocculation/precipitation, to the fermentation broth supernatant prior to membrane filtration, or to the fermentation product after membrane filtration.

We have found that fungal dnase can be advantageously applied to the fermentation product after membrane filtration which reduces the amount of water and other low molecular compounds (ultrafiltration) thereby increasing the concentration of the protein of interest and residual host cell DNA.

According to the invention, when a fungal dnase or variant thereof is "added" or "administered" this excludes (endogenous) production/expression of the dnase by the microbial host cell in the fermentation broth. Fungal dnases are isolated or recovered enzymes added or applied from an external source. Administration of dnase from an external source is advantageous because the production capacity of the microbial host cell is then exclusively used for the production of (commercial) proteins of interest.

DNA is considered to be removed when it degrades into individual nucleotides or oligonucleotides of less than 150bp, for example as measured by digital PCR.

Microbial host cells

The microbial host cell may be a host cell of any genus. The desired protein of interest may be homologous or heterologous to the host cell capable of producing the protein of interest.

The term "homologous protein" or "native protein" means a protein encoded by a gene derived from the host cell in which it is produced.

The term "heterologous protein" means a protein encoded by a gene that is foreign to the host cell in which it is produced.

As used herein, the term "recombinant host cell" means a host cell that comprises a gene(s) encoding a desired protein and is capable of expressing the gene(s) to produce the desired protein. The desired protein-encoding gene(s) may be introduced into the recombinant host cell via transformation, transfection, transduction, etc., using techniques well known in the art.

When the desired protein is a heterologous protein, the recombinant host cell capable of producing the desired protein is preferably of fungal or bacterial origin. The choice of recombinant host cell will depend to a large extent on the gene encoding the desired protein and the source of the protein.

As used herein, the term "wild-type host cell" refers to a host cell that naturally comprises the gene(s) encoding the desired protein and is capable of expressing the gene(s).

The mutant may be a wild-type host cell in which one or more genes have been deleted (e.g., to enrich for the desired protein preparation).

In a preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium or fungus.

The microbial host cell may be a yeast cell, such as a Candida (Candida), hansenula (Hansenula), kluyveromyces (Kluyveromyces), pichia (Pichia), saccharomyces (Saccharomyces), schizosaccharomyces (Schizosaccharomyces), or Yarrowia (Yarrowia) strain. In another aspect, the strain is a strain of Saccharomyces cerevisiae (Saccharomyces carlsbergensis), saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces diastaticus (Saccharomyces diastaticus), saccharomyces cerevisiae (Saccharomyces douglasii), kluyveromyces (Saccharomyces kluyveri), saccharomyces noris (Saccharomyces norbensis), or Saccharomyces ovale (Saccharomyces oviformis).

The microbial host cell may be a filamentous fungal strain, such as Acremonium (Acremonium), agaricus (Agaricus), alternaria (Alternaria), aspergillus (Aspergillus), aureobasidium (Aureobasidium), portugal (Botryospaeria), ceriporiopsis (Ceriporiopsis), mao Hui crust (Chaetomidium), chrysosporium (Chrysosporium), clavipita (CLAVICEPS), alternaria (Cochliobolus), coprinus (Coprinopsis), alternaria (Coptotermes), alternaria (Corynascus), cryptococcus (Cryphonectria), cryptococcus (Cryptococcus), saccharomyces (Diplodia), heterocarpa (Exidia), brevibacterium (Filibalium), fusarium (Samerium), gibbella (Gibbella), fuscoporia (Fubbella) whole flagella (Holomastigotoides), humicola (Humicola), harringtoni (Irpex), lentinula (Lentinula), micrococcus (Leptospaeria), pyriform fungus (Magnaporthe), black fruit fungus (Melanocarpus), grifola (Meripilus), mucor (Mucor), myceliophthora (Myceliophthora), new mechnia (neocalimagax), neurospora (Neurospora), paecilomyces (Paecilomyces), penicillium (Penicillium), phanerochaete (Phanerochaete), ruminococcus (Piromyces), poitrasia, pseudoblack dish fungus (Pseudoplectania), pseudo with dishevelled hair pest (Pseudotrichonympha), rhizomucor), schizophyllum (Schizophyllum), strains of Acremonium (Scytalidium), bryonia (Talaromyces), thermoascus (Thermoascus), thielavia (Thielavia), tolypocladium (Tolypocladium), trichoderma (Trichoderma), pantoea (Trichophaea), verticillium (Verticillium), pachymopapain (Volvariella), or Xylobacter (Xylaria).

In another aspect, the strain is Acremonium cellulolyticus (Acremonium cellulolyticus), aspergillus aculeatus (Aspergillus aculeatus), aspergillus awamori (Aspergillus awamori), aspergillus foetidus (Aspergillus foetidus), aspergillus fumigatus (Aspergillus fumigatus), aspergillus japonicus (Aspergillus japonicus), aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), chrysosporium angustifolium (Chrysosporiuminops), chrysosporium keratinophilum (Chrysosporium keratinophilum), lu Kenuo Trichosporon fraxinum (Chrysosporium lucknowense), chrysosporium faecalis (Chrysosporium merdarium), chrysosporium rententium (Chrysosporium pannicola), Goldschia kunmingensis (Chrysosporium queenslandicum), chrysosporium tropicalis (Chrysosporium tropicum), chrysosporium striatum (Chrysosporium zonatum), fusarium culmorum (Fusarium bactridioides), fusarium cereal (Fusarium cerealis), fusarium kuweise (Fusarium crookwellense), fusarium culmorum (Fusarium culmorum), Fusarium graminearum (Fusarium graminearum), fusarium graminearum (Fusarium graminum), fusarium heterosporum (Fusarium heterosporum), fusarium negundo (Fusarium negundi), fusarium oxysporum (Fusarium oxysporum), fusarium polycephalum (Fusarium reticulatum), fusarium roseum (Fusarium roseum), fusarium sambucinum (Fusarium sambucinum), fusarium sambucinum, Fusarium skin color (Fusarium sarcochroum), fusarium pseudomycoides (Fusarium sporotrichioides), fusarium sulphureum (Fusarium sulphureum), fusarium toruloides (Fusarium torulosum), fusarium pseudosilk (Fusarium trichothecioides), fusarium venenatum (Fusarium venenatum), humicola grisea, humicola insolens (Humicola insolens), humicola insolens (Fusarium oxysporum), Humicola lanuginosa (Humicola lanuginosa), rake fungus (Irpex lacteus), mucor miehei (Mucor miehei), myceliophthora thermophila (Myceliophthora thermophila), streptomyces crassipes (Neurospora crassa), penicillium funiculosum (Penicillium funiculosum), penicillium purpurogenum (Penicillium purpurogenum), phanerochaete chrysosporium (Phanerochaete chrysosporium), Thielavia leucotrichum (THIELAVIA ACHROMATICA), thielavia layering (THIELAVIA ALBOMYCES), thielavia Bai Maosuo (THIELAVIA ALBOPILOSA), thielavia australis (THIELAVIA AUSTRALEINSIS), thielavia fei (THIELAVIA FIMETI), thielavia minima (THIELAVIA MICROSPORA), thielavia ootheca (THIELAVIA OVISPORA), Thielavia Peruvialis (THIELAVIA PERUVIANA), thielavia terrestris (THIELAVIA SETOSA), thielavia lanuginosus (THIELAVIA SPEDEDONIUM), thielavia thermophilus (THIELAVIA SUBTHERMOPHILA), thielavia terrestris (THIELAVIA TERRESTRIS), trichoderma harzianum (Trichoderma harzianum), trichoderma koningii (Trichoderma koningii), trichoderma longibrachiatum (Trichoderma longibrachiatum), trichoderma reesei (Trichoderma reesei), or Trichoderma viride (Trichoderma viride).

In one aspect, the fungal host cell is a strain selected from the group consisting of: candida, hansenula, kluyveromyces, pichia, saccharomyces, schizosaccharomyces, yarrowia, acremonium, aspergillus, fusarium, humicola, mucor, myceliophthora, neurospora, penicillium, fusheca, torticola, and trichoderma.

In a more preferred embodiment, the filamentous fungal host cell is selected from the group consisting of: trichoderma and Aspergillus host cells, in particular Trichoderma harzianum, trichoderma koningii, trichoderma longibrachiatum, trichoderma reesei, trichoderma viride, aspergillus awamori, aspergillus fumigatus, aspergillus foetidus, aspergillus japonicus, aspergillus nidulans, aspergillus niger or Aspergillus oryzae strains, in particular Trichoderma reesei strains.

In another preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium.

The recombinant host cell may comprise a single copy or at least two copies, e.g., three, four, five or more copies, of a polynucleotide of the invention.

The host cell may be any gram positive or gram negative bacterium. Gram positive bacteria include, but are not limited to: bacillus, clostridium, enterococcus, geobacillus, lactobacillus, lactococcus, bacillus, staphylococcus, streptococcus and streptomyces. Gram negative bacteria include, but are not limited to: campylobacter, escherichia coli, flavobacterium, fusobacterium, helicobacter, mudacter, neisseria, pseudomonas, salmonella, and ureaplasma.

The host cell may be any Bacillus cell including, but not limited to, bacillus alcalophilus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), bacillus circulans (Bacillus circulans), bacillus clausii (Bacillus clausii), bacillus coagulans (Bacillus coagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus subtilis (Bacillus subtilis), and Bacillus thuringiensis (Bacillus thuringiensis) cells. In one embodiment, the bacillus cell is a bacillus amyloliquefaciens, bacillus licheniformis, or bacillus subtilis cell.

In one embodiment, the bacillus cell is a bacillus subtilis cell.

In another embodiment, the bacillus cell is a bacillus licheniformis cell.

For the purposes of the present invention, bacillus species/genus/species shall be defined as described in Patel and Gupta,2020, int.J.Syst.Evol.Microbiol. [ J.International System and evolutionary microbiology ] 70:406-438.

The bacterial host cell may also be any streptococcus cell including, but not limited to, streptococcus equisimilis (Streptococcus equisimilis), streptococcus pyogenes (Streptococcus pyogenes), streptococcus uberis (Streptococcus uberis) and streptococcus equi subsp.

The bacterial host cell may also be any Streptomyces cell including, but not limited to, streptomyces diastatochromogenes, streptomyces avermitilis, streptomyces coelicolor, streptomyces griseus, and Streptomyces lividans cells.

Methods for introducing DNA into prokaryotic host cells are well known in the art and any suitable method may be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. One skilled in the art will be readily able to determine the appropriate method for introducing DNA into a given prokaryotic cell, depending on, for example, genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al, 2018,BMC Microbiology[BMC microbiology [ 18:56 ], burke et al, 2001, proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]98:6289-6294, choi et al, 2006, J. Microbiol. Methods [ J. Methods of microorganisms ]64:391-397, and Donald et al, 2013, J. Bacteriol. [ J. Bacteriology ]195 (11): 2612-2620.

Fungal DNase

The fungal dnase used in the present invention is a deoxyribonuclease derived from a fungal microorganism or a variant thereof. DNase is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. There are two main classifications depending on the site of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of DNA molecules.

Examples of fungal dnases can be found, for example, in patent applications WO 2017/059802 and WO 2017/059801 (incorporated by reference), which disclose amino acid sequences encoding fungal dnases. Fungal dnase is a wild-type dnase derived from a fungal strain. A particularly preferred fungal DNase is Aspergillus oryzae DNase as shown in SEQ ID NO. 2.

Variants of fungal dnase may have more than 60%, more than 70%, more than 80% or more than 90% amino acid sequence identity with fungal wild-type dnase.

In a particular embodiment, the fungal DNase or variant thereof has more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% amino acid sequence identity with SEQ ID NO. 2. Alternatively, the fungal dnase or variant thereof has 1,2,3, 4 or 5 amino acid substitutions, deletions or insertions, preferably 1,2,3, 4 or 5 conservative amino acid substitutions compared to SEQ ID No. 2.

In a preferred embodiment, the fungal dnase or variant thereof is NUC3 nuclease.

The subgroup of DNase_NucA_ NucB (Pfam domain id PF14040, pfam version 31.0Finn (2016). Nucleic ACIDS RESEARCH [ Nucleic acids research ], database 44 th stage: D279-D285) is referred to as NUC3. The NUC3 nuclease comprises a dnase_nuca_ NucB domain and comprises any one of the motif [ LV ] [ PTA ] [ FY ] [ DE ] [ VAGPH ] D [ CFY ] [ WY ] [ AT ] [ IM ] L [ CYQ ] corresponding to amino acids 24 to 35 in an aspergillus oryzae dnase having the amino acid sequence shown in SEQ ID No. 2 and/or the motif GPYCK (SEQ ID NO: 3) corresponding to amino acids 157 to 161 in an aspergillus oryzae dnase having the amino acid sequence shown in SEQ ID No. 2 or the motif WF [ QE ] IT (SEQ ID NO: 4) corresponding to amino acids 146 to 150 in an aspergillus oryzae dnase having the amino acid sequence shown in SEQ ID No. 2.

Thus, the A.oryzae DNase shown in SEQ ID NO. 2 is a NUC3 DNase.

As described above, amino acid changes in dnase variants may have minor properties, i.e. conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxy-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function (such as a polyhistidine segment, epitope, or binding moiety).

Essential amino acids in polypeptides (proteins, enzymes) can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,1989, science [ science ] 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the resulting molecule is tested for dnase activity to identify amino acid residues critical to the activity of the molecule. See also Hilton et al, 1996, J.biol.chem. [ J.Biochem. ]271:4699-4708. The active site of an enzyme or other biological interaction may also be determined by physical analysis of the structure, as determined by techniques such as: nuclear magnetic resonance, crystallography (crystallography), electron diffraction, or photoaffinity labeling, along with mutating putative contact site amino acids. See, e.g., de Vos et al, 1992, science [ science ]255:306-312; smith et al, 1992, J.mol.biol. [ J.Mol.Biol. ]224:899-904; wlodaver et al, 1992, FEBS Lett. [ European society of Biochemical Association flash ]309:59-64. The identity of essential amino acids can also be deduced from an alignment with the relevant polypeptide and/or from sequence homology and conserved catalytic mechanisms within the relevant polypeptide or protein family with polypeptides/proteins from a common ancestor (typically having similar three-dimensional structure, function and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used in protein structure modeling to identify essential amino acids and/or active sites of polypeptides. See, e.g., jumper et al 2021, "Highly accurate protein structure prediction with AlphaFold [ highly accurate protein structure predictions using alpha folding ]", nature [ Nature ]596:583-589.

Single or multiple amino acid substitutions, deletions and/or insertions may be made and tested using known mutagenesis, recombination and/or shuffling methods followed by related screening procedures such as those described by Reidhaar-Olson and Sauer,1988, science [ science ]241:53-57; bowie and Sauer,1989, proc.Natl. Acad.Sci.USA [ Proc. Natl. Acad. Sci. USA, U.S. national academy of sciences ]86:2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that may be used include error-prone PCR, CRISPR gene editing, phage display (e.g., lowman et al, 1991, biochemistry [ biochemistry ]30:10832-10837;US 5,223,409;WO 92/06204), and region-directed mutagenesis (Derbyshire et al, 1986, gene [ gene ]46:145; ner et al, 1988, DNA 7:127).

Protein of interest

The protein of interest is produced by a microbial host cell. Such proteins may be small (peptide, <50 amino acids) or large (polypeptide, >50 amino acids) biomolecules that perform a variety of functions within living organisms, including catalytic reactions, DNA replication, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins are composed of polymeric amino acid chains that fold in a very specific three-dimensional structure. The three-dimensional structure is critical to maintaining the function of the protein. Some chemicals can alter folding and even unfold (denature) the three-dimensional structure, which will lead to loss of function, e.g. loss of enzymatic activity.

In one embodiment, the protein is a polypeptide; preferably globulin/polypeptide. In another embodiment, the protein is soluble under physiological conditions.

Proteins fall into at least four distinct groups, namely enzymes, cell signaling proteins, ligand binding proteins, and structural proteins.

In another embodiment, at least one polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of: antibodies, antibody fragments, antibody-based drugs, fc fusion proteins, anticoagulants, blood factors, bone morphogenic proteins, engineered protein scaffolds, growth factors, clotting factors, hormones, interferons (e.g., interferon alpha-2 b), interleukins, lactoferrin, alpha-lactalbumin, beta-lactalbumin, ovomucoid, ovo-solid, cytokines, adiponectin, human galactosidase (e.g., human alpha-galactosidase a), vaccines, protein vaccines, and thrombolytics.

Enzymes are described below.

Cell signaling proteins and ligand binding proteins include proteins such as, for example, receptors, membrane proteins, ion channels, antibodies (e.g., single domain antibodies), hormones, hemoglobin, and hemoglobin-like molecules. Heme-containing enzymes, such as peroxygenases and peroxidases, may comprise an enzymatic activity, or may be variants of an inactivated heme-containing enzyme.

Structural proteins provide stiffness and rigidity to other fluid biological components.

Preferably, the protein is an enzyme, a cell signaling protein or a ligand binding protein; more preferably, the protein is an enzyme.

Enzymes

The protein of interest may be an enzyme (catalytic protein). When the protein is an enzyme, the amount of protein is an active enzyme protein.

The term "active enzyme protein" is defined herein as the amount of one or more catalytic proteins that exhibit enzymatic activity. This can be determined using an activity-based analytical enzyme assay. In such assays, enzymes typically catalyze reactions that produce colored compounds. The amount of colored compound can be measured and correlated to the concentration of active enzyme protein. This technique is well known in the art.

The one or more enzymes may be one or more enzymes, for example selected from the group consisting of: hydrolytic enzymes, lytic enzymes, transferases, proteases, amylases, glucoamylases, pectinases, pectin lyases, cellulases, xylanases, arabinanases, arabinofuranosidases, mannanases, carrageenases, xanthanases, endoglucanases, chitinases, asparaginases, lipases, phospholipases, cutinases, lysozyme, phytases, deamidases, transglutaminases, oxidoreductases (e.g., carbohydrate oxidases, laccases, peroxidases, catalytic enzymes), lactases, glucose isomerases, xylose isomerases, and esterases.

The enzyme may be an enzyme of naturally occurring bacterial or fungal origin, or it may be a variant derived from one or more naturally occurring enzymes by gene shuffling and/or by substitution, deletion or insertion of one or more amino acids. Chemically modified mutants or protein engineered mutants are included.

Fermentation liquor

The invention may be useful for any fermentation on an industrial scale, for example for any fermentation having at least 50 litres, preferably at least 500 litres, more preferably at least 5,000 litres, even more preferably at least 50,000 litres of medium.

Microorganisms producing the protein of interest may be fermented by any method known in the art. The fermentation medium may be a minimal medium as described in, for example, WO 98/37179, or the fermentation medium may be a complex medium comprising a complex nitrogen source and a carbon source, wherein the complex nitrogen source may be partially hydrolysed as described in WO 2004/003216.

The fermentation may be performed in a batch, repeated batch, fed-batch, repeated fed-batch, or continuous fermentation process.

In a fed-batch process, no compound comprising one or more nutrients is added or a portion thereof is added to the medium prior to starting the fermentation, and correspondingly, all or the remaining portion of the compound comprising one or more nutrients is fed during the fermentation. The compounds selected as feed may be fed together or separately to the fermentation process.

In repeated fed-batch or continuous fermentation processes, the complete starter medium is additionally fed during the fermentation. The starting medium may be fed in together with the structural element feed(s) or separately. In a repeated fed-batch process, a portion of the fermentation broth comprising biomass is removed at regular time intervals, whereas in a continuous process, the removal of a portion of the fermentation broth is performed continuously. Thereby, the fermentation process is supplemented with a portion of fresh medium corresponding to the withdrawn amount of fermentation broth.

In a preferred embodiment of the invention, fermentation broth from a fed-batch fermentation process is preferred.

In one embodiment, the fermentation broth is provided after a incubation time of at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

In a particular embodiment, the fermentation broth is provided after a incubation time of at least 120 hours.

According to the invention, the fermentation broth may be diluted to 2000% (w/w) with water; preferably, the fermentation broth may be diluted with water by 10% -2000% (w/w); more preferably, the fermentation broth may be diluted 100% -1500% (w/w) with water; more preferably, the fermentation broth may be diluted 100% -1000% (w/w) with water; more preferably, the fermentation broth may be diluted 200% -800% (w/w) with water.

According to the invention, dilution with water means that the dilution medium may be water, or it may be ultrafiltration permeate from the production of the protein of interest, or it may be recycled water from the production of the protein of interest, or it may be condensate from a heater, or it may be any combination of the above mentioned, such as a mixture of water and ultrafiltration permeate.

The fermentation broth comprises host cells (including host cells comprising a gene encoding a polypeptide of interest, which is used to produce the polypeptide of interest), cell debris, biomass, recombinant DNA from bacterial host cells, fermentation medium, and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing one or more organic acids, killed cells and/or cell debris, and culture medium.

For example, when a microbial culture is grown to saturation under carbon-limiting conditions that allow protein synthesis (e.g., expression of enzymes by a host cell) and secretion of the protein into the cell culture medium, a fermentation broth is produced. The fermentation broth may contain the unfractionated or fractionated content of the fermentation material derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent medium and cell debris present after removal of microbial cells (e.g., bacillus cells), such as by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.

The cell-killed whole culture broth or cell composition may contain the unfractionated contents of the fermentation material derived at the end of the fermentation. Typically, the cell-killing whole culture fluid or cell composition contains spent medium and cell debris present after microbial cells (e.g., bacillus cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole culture fluid or cell composition contains spent cell culture medium, recombinant DNA from microbial host cells, extracellular enzymes, and killed microbial cells. In some embodiments, methods known in the art may be used to permeabilize and/or lyse microbial cells present in a cell-killing whole culture or composition.

The whole culture fluid or cell composition as described herein is typically a liquid, but may contain insoluble components such as killed cells, recombinant DNA from microbial host cells, cell debris, media components, and/or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

The whole culture broth formulation and cell composition of the invention may be produced by the methods described in WO 90/15861 or WO 2010/096673.

Flocculation/precipitation

To flocculate the fermentation broth, divalent salts may be added to the fermentation broth, in particular calcium and/or magnesium salts, for example calcium chloride or magnesium chloride. Preferred embodiments are calcium salts, in particular calcium chloride.

The salt may be added to the fermentation broth at a concentration of 0.01% -10% (w/w)/kg fermentation broth (undiluted), preferably 0.5% -10% (w/w)/kg fermentation broth (undiluted), more preferably 1% -9% (w/w)/kg fermentation broth (undiluted), in particular 2% -8% (w/w)/kg fermentation broth (undiluted).

Polyaluminium compounds

To further improve the removal of DNA from the fermentation broth, a polyaluminium compound may be added to the fermentation broth. A wide variety of aluminum compounds are known to improve flocculation, such as ,Al₂(SO₄)₃、NaAlO₂、K₂Al₂O₄、AlCl₃、Al(NO₃)₃、Al- acetate, and Al-formate.

Particularly useful polyaluminium chlorides include compounds having the formula Al _n(OH)_mCl(_3n-m) and polyaluminium chlorides and aluminum hydroxychloride (aluminium chlorohydrate) having CAS numbers 1327-41-9.

Examples of useful polyaluminum chlorides include aluminum hydroxychloride (aluminum chlorohydrate), GC850 ^TM(Al₂(OH)₅ Cl available from Goulbrandsen, inc., or NordPac 18 (available from No Wo Nuo g company (Nordisk Aluminat A/S), denmark), which is an aluminum complex having the empirical formula (brutto formula) Al (OH) _1,2Cl_1,8. Another example of a useful polyaluminum chloride having the formula Al (OH) _1,2Cl_1,8 is PAX-XL 100 (available from Kemira). Two other examples of useful polyaluminium chlorides are PAC (available from Shanghai water treatment facilities limited (Shanghai Haotian WATER TREATMENT Equipment co., ltd), supplied in solid form) or PAC (available from Kaiser technology limited (Tianjin Kairuite technology Ltd), supplied in liquid form). Another example of a useful polyaluminum chloride having the formula Al (OH) _1,2Cl_1,8 is PAX18 (available from Kemira water treatment company (KEMIRA WATER Solutions)).

The concentration of polyaluminum chloride will typically be in the range of 0.1% -10% (w/w) per kg of fermentation broth (undiluted); preferably in the range of 0.5% -5% (w/w) per kg of fermentation broth (undiluted).

After the addition of polyaluminum chloride, the pH may be adjusted. The pH may be adjusted to a pH in the range of pH 2 to pH 11. The pH may be adjusted with any acid or base known in the art.

Polyaluminum chloride may also be added after the microorganism has been separated from the fermentation broth.

Polyaluminum chloride may also be added in two or more steps: for example, prior to removing microorganisms from the fermentation broth; and then added again after the microorganisms have been removed, for example in a subsequent downstream process liquid.

Polymer

The polymer may be used for particle agglomeration. Anionic and cationic polymers are preferred. Useful cationic polymers may be polyamines and useful anionic polymers may be polyacrylamides. Useful polymer concentrations typically range from 0.5% to 20% (w/w) per kg of fermentation broth (undiluted); preferably in the range of 1% -10% (w/w) per kg of fermentation broth (undiluted).

An example of a useful anionic polymer is Superfloc ^TM A130 (Kemira). Examples of useful cationic polymers are Polycat ^TM (Ke Mira Co.), C521 (Ke Mira Co.), and C591 (Ke Mira Co.).

Filtration and other downstream operations

Flocculated cell debris may be removed by methods known in the art, such as, but not limited to, filtration, e.g., drum filtration, membrane filtration, filter press terminal filtration, cross-flow filtration, or centrifugation.

The resulting fermentation supernatant may then be further processed or refined by methods known in the art. For example, the protein may be recovered by conventional procedures including, but not limited to, further filtration (e.g., ultrafiltration and diafiltration), extraction, spray drying, evaporation, precipitation, or crystallization.

The term "recovery" or "recovery" means removing a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, e.g., recovering the polypeptide from the whole fermentation broth or from a cell-free fermentation broth by: polypeptide is harvested from broth by polypeptide crystal harvesting, by filtration (e.g., by filtration using filter aid or fill filter media, cloth filtration in a box filter, drum filtration, rotary vacuum drum filtration, candle filters, horizontal leaf filters or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, crossflow, dynamic crossflow or ultrafiltration in dead-end operation)) or by centrifugation (using a horizontal centrifuge, disk stack centrifuge, hydrocyclone or the like) or by precipitation of polypeptide and using related solid-liquid separation methods to harvest polypeptide from broth by using particle size fractionation. Recovery encompasses isolation and/or purification of polypeptides.

The isolated protein may then be further purified and/or modified using various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography) and/or electrophoretic procedures (e.g., preparative isoelectric focusing electrophoresis) and/or solubility differences (e.g., ammonium sulfate precipitation) and/or extraction.

Microfiltration may involve membranes with the following size exclusion limits: greater than 1000kDa, greater than 500kDa, greater than 100kDa, or greater than 50kDa; and/or greater than 5 μm, greater than 1 μm, greater than 0.5 μm, greater than 0.4 μm, greater than 0.3 μm, greater than 0.2 μm, or greater than 0.1 μm; or other filters having equivalent molecular weight exclusion properties; wherein the fermentation product is a microfiltration filtrate.

After microfiltration, or in a combined process, the fermentation product may undergo an ultrafiltration step involving a membrane having a size exclusion limit of greater than 100kDa, greater than 80kDa, greater than 60kDa, greater than 50kDa, greater than 40kDa, greater than 30kDa, greater than 20kDa, greater than 15kDa, greater than 10kDa, greater than 5kDa, or greater than 1 kDa; or another filter having equivalent molecular weight exclusion properties.

The microbial fermentation product may be a liquid or solid formulation, or it may be used to prepare such a formulation.

The liquid formulation may comprise a polyol (polyol/polyhydric alcohol), for example in an amount of at least 10% w/w, at least 25% w/w or at least 50% w/w. The polyol is an alcohol having two or more hydroxyl groups. Useful polyols typically have molecular weights below 500 g/mol.

Polyols include non-saccharide polyols such as glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), and sugar alcohols such as sorbitol, mannitol, erythritol, galactitol, inositol, xylitol, and ribitol. Polyols also include sugar polyols, such as mono-and disaccharides, e.g., glucose, fructose, galactose, sucrose, lactose, maltose and trehalose.

The solid formulation may be granules prepared by, for example, high shear granulation or fluid bed granulation or a combination. Coatings with one or more salts or one or more polymers may also be applied.

Detection of residual host cell DNA

The term residual host cell DNA includes genomic DNA from the production strain and DNA fragments encoding the protein of interest.

Detection and quantification of trace amounts of residual host cell DNA can be accomplished by various methods known in the art. Numerous methods have been developed to determine specific individual target sequences. The method comprises the following steps:

(a) Hybridization-based methods for detecting specific DNA of defined origin using dot blotting, hybridization of radioisotope-labeled DNA probes using random hexamers to generate representative probes covering the whole genome of these host cells;

(b) A quantitative PCR-based method for detecting specific DNA of defined origin that targets specific gene sequences for amplification and calibration using purified, species-matched, genomic DNA; and

(C) Qualitative PCR methods for detecting specific DNA of defined origin that targets specific gene sequences for amplification and calibration using purified, species-matched, genomic DNA. The threshold is determined based on the lowest amount of genomic DNA that can be detected using this method.

According to the present invention, purification of trace amounts of DNA was accomplished by using FastDNA ^TM Spin kit (Ansebiological medical (MP Biomedicals)). The eluted sample is then subjected to a PCR reaction using specific primers directed to chromosomal loci on the host cells. Positive controls were included in which known amounts of host DNA were added to the PCR reaction at different concentrations. After the PCR reaction, the samples were subjected to gel electrophoresis and the intensities of the DNA bands were compared in order to estimate the concentration of host DNA in the initial sample. The detailed protocol for PCR is provided below: lnnis et al (1990) PCR ProtocoIs, A Guide to methods and applications [ PCR protocol: methods and application guidelines ], ACADEMIC PRESS INC [ academic press company ], new york.

Treatment of a fermentation broth or other protein formulation using the present method results in a significant reduction in the amount of DNA present in the fermentation broth, and preferably, the DNA content is reduced to undetectable levels. Levels are considered undetectable if PCR amplification of any segment of genomic DNA present as a single copy in the haploid genome gives no visible band after ethidium bromide staining.

Preferably, the DNA level in the fermentation broth is reduced to a level below 1. Mu.g/ml, preferably below 500ng/ml, preferably below 200ng/ml, preferably below 100ng/ml, preferably below 50ng/ml, preferably below 20ng/ml, preferably below 10ng/ml, preferably below 5ng/ml, preferably below 2ng/ml, preferably below 1ng/ml, and most preferably below 500pg/ml.

In one embodiment, the microbial fermentation product comprises less than 10ng/g, less than 5ng/g, less than 1ng/g, less than 0.5ng/g, less than 0.1ng/g, less than 0.05ng/g, or less than 0.01ng/g recombinant DNA.

In one example of a typical regulatory environment, for example, at a detection limit of 1, 5, 10, or 20ng/mL enzyme preparation, no detectable DNA can be determined using a PCR-based assay.

In addition, the use of the instant method in combination with conventional methods of removing DNA from fermentation broths or other protein preparations is also contemplated.

Some further embodiments of the invention include:

example 1A method for reducing the amount of DNA in a microbial fermentation product comprising

wherein the fungal dnase or variant thereof is added to the fermentation medium before or during fermentation, to the fermentation broth after or in step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c).

Embodiment 2. The method of embodiment 1, wherein the fungal dnase or variant thereof is added to the fermentation medium before or during fermentation.

Example 3. The method of example 1, wherein the fungal dnase or variant thereof is added to the fermentation broth in step (a).

Example 4. The method of example 1, wherein the fungal dnase or variant thereof is added to the fermentation broth after step (a).

Example 5. The method of example 1, wherein the fungal dnase or variant thereof is added to the fermentation broth after step (b).

Example 6. The method of example 1, wherein the fungal dnase or variant thereof is added to the fermentation broth after step (c).

Embodiment 7. The method of any one of the preceding embodiments, wherein the protein of interest is heterologous to the microbial host cells.

Embodiment 8. The method of any one of the preceding embodiments, wherein the protein of interest is secreted into the fermentation broth by the microbial host cells.

Embodiment 9. The method of any one of the preceding embodiments, wherein the protein of interest is not secreted into the fermentation broth by the microbial host cells.

Embodiment 10. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 0.1% w/w.

Embodiment 11. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 0.5% w/w.

Embodiment 12. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 1% w/w.

Embodiment 13. The method of any one of the preceding embodiments, wherein the protein of interest is an enzyme, a heme-containing protein, a cell signaling protein, or a ligand binding protein.

Embodiment 14. The method of any one of the preceding embodiments, wherein the protein of interest is an enzyme.

Embodiment 15. The method of any of the preceding embodiments, wherein the membrane filtration in step (c) comprises a microfiltration step.

Embodiment 16. The method of the preceding embodiment, wherein the microfiltration membrane has a size exclusion limit of 0.1 μm to 10 μm.

Embodiment 17. The method of the preceding embodiment, wherein the microfiltration membrane has a size exclusion limit of 0.5 μm to 5 μm.

Embodiment 18. The method of any of the preceding embodiments, wherein the membrane filtration in step (c) comprises an ultrafiltration step.

Embodiment 19. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 100 kDa.

Embodiment 20. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 50 kDa.

Embodiment 21. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 40 kDa.

Embodiment 22. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 30 kDa.

Embodiment 23. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 20 kDa.

Embodiment 24. The method of the preceding embodiment, wherein the ultrafiltration membrane has a size exclusion limit of less than 10 kDa.

Embodiment 25. The method of any one of the preceding embodiments, wherein some or all of the microbial host cells in step (a) have been mechanically or enzymatically disrupted/homogenized.

Embodiment 26. The method of any of the preceding embodiments, wherein the fungal dnase or variant thereof is a wild-type fungal dnase or a variant thereof having at least 60% amino acid sequence identity with the wild-type fungal dnase, and wherein the variant exhibits dnase activity.

Embodiment 27. The method of embodiment 25, wherein the variant has at least 70% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 28. The method of embodiment 25, wherein the variant has at least 80% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 29. The method of embodiment 25, wherein the variant has at least 90% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 30. The method of embodiment 25, wherein the variant has at least 95% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 31. The method of embodiment 25, wherein the variant has at least 96% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 32. The method of embodiment 25, wherein the variant has at least 97% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 33. The method of embodiment 25, wherein the variant has at least 98% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 34. The method of embodiment 25, wherein the variant has at least 99% amino acid sequence identity to the wild-type fungal dnase.

Embodiment 35. The method of any one of the preceding embodiments, wherein the fungal dnase or variant thereof is a wild-type fungal dnase or variant thereof having 1,2, 3, 4, or 5 substitutions, deletions, or insertions, preferably 1,2, 3, 4, or 5 conservative substitutions.

Embodiment 36. The method of any one of the preceding embodiments, wherein the fungal dnase or variant thereof is a wild-type fungal dnase or variant thereof having 1,2,3,4 or 5 conservative substitutions.

Embodiment 37 the method of any one of embodiments 25-35, wherein the wild-type fungal dnase has an amino acid sequence as set forth in SEQ ID No. 2.

Embodiment 38. The method of any one of the preceding embodiments, wherein the fungal dnase or variant thereof is NUC3 nuclease.

Embodiment 39. The method of any of the preceding embodiments, wherein the fungal dnase or variant thereof comprises the dnase_nuca_ NucB domain and further comprises any of the motifs [ LV ] [ PTA ] [ FY ] [ DE ] [ VAGPH ] D [ CFY ] [ WY ] [ AT ] [ IM ] L [ CYQ ] and/or the motifs GPYCK (SEQ ID NO: 3) or WF [ QE ] IT (SEQ ID NO: 4).

Embodiment 40. The method of any one of the preceding embodiments, wherein the fungal dnase or variant thereof comprises or consists of the amino acid sequence of SEQ ID No. 2.

Embodiment 41 the method of any one of the preceding embodiments, wherein the microbial host cell is a bacterial host cell, a fungal host cell, or a yeast host cell; preferably, the microbial host cell is a bacterial host cell.

Embodiment 42. The method of any of the preceding embodiments, wherein the microbial host cell is a strain selected from the group consisting of: candida, hansenula, kluyveromyces, pichia, saccharomyces, schizosaccharomyces, yarrowia, acremonium, aspergillus, fusarium, humicola, mucor, myceliophthora, neurospora, penicillium, fusheca, torticola, and trichoderma.

Embodiment 43 the method of any one of the preceding embodiments, wherein the microbial host cell is a strain of pichia.

Embodiment 44 the method of any one of the preceding embodiments, wherein the microbial host cell is a strain of Pichia pastoris.

Embodiment 45. The method of any of the preceding embodiments, wherein the microbial host cell is a strain selected from the group consisting of: bacillus, streptomyces, escherichia, brucella (Buttiauxella) and Pseudomonas.

Embodiment 46. The method of any one of the preceding embodiments, wherein the microbial host cell is a strain of bacillus or escherichia.

Embodiment 47 the method of any one of the preceding embodiments, wherein the microbial host cell is a bacillus host cell; preferred are Bacillus amyloliquefaciens, bacillus licheniformis, or Bacillus subtilis host cells.

Embodiment 48. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 10ng/g recombinant DNA.

Embodiment 49. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 5ng/g recombinant DNA.

Embodiment 50. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 1ng/g recombinant DNA.

Embodiment 51. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.5ng/g recombinant DNA.

Embodiment 52. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.1ng/g recombinant DNA.

Embodiment 53. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.05ng/g recombinant DNA.

Embodiment 54. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.01ng/g recombinant DNA.

Embodiment 55. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises the fungal dnase or variant thereof.

Example 56A method for reducing the amount of DNA in a microbial fermentation product comprising

Wherein dnase is added to the fermentation medium before or during fermentation, to the fermentation broth in or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c);

wherein the dnase has at least 80% amino acid sequence identity with SEQ ID No. 2; and

Wherein the microbial fermentation product comprises less than 10ng/g recombinant DNA.

Embodiment 57. The method of embodiment 56, wherein the DNase is added to the fermentation medium before or during fermentation.

Example 58 the method of example 56, wherein the dnase is added to the fermentation broth in step (a).

Example 59 the method of example 56, wherein the dnase is added to the fermentation broth after step (a).

Example 60. The method of example 56, wherein the dnase is added to the fermentation broth after step (b).

Example 61 the method of example 56, wherein the dnase is added to the fermentation broth after step (c).

Embodiment 62. The method of any of the preceding embodiments, wherein the protein of interest is heterologous to the microbial host cells.

Embodiment 63. The method of any of the preceding embodiments, wherein the protein of interest is secreted into the fermentation broth by the microbial host cells.

Embodiment 64. The method of any of the preceding embodiments, wherein the protein of interest is not secreted into the fermentation broth by the microbial host cells.

Embodiment 65. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 0.1% w/w.

Embodiment 66. The method of any one of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 0.5% w/w.

Embodiment 67. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises a protein of interest in an amount of at least 1% w/w.

Embodiment 68. The method of any of the preceding embodiments, wherein the protein of interest is an enzyme, a heme-containing protein, a cell signaling protein, or a ligand binding protein.

Embodiment 69. The method of any one of the preceding embodiments, wherein the protein of interest is an enzyme.

Embodiment 70. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 50 kDa.

Embodiment 71. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 40 kDa.

Embodiment 72. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 30 kDa.

Embodiment 73. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 20 kDa.

Embodiment 74. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 10 kDa.

Embodiment 75. The method of any one of the preceding embodiments, wherein some or all of the microbial host cells in step (a) have been mechanically or enzymatically disrupted/homogenized.

Embodiment 76. The method of any of the preceding embodiments, wherein the dnase has at least 85% amino acid sequence identity with SEQ ID No. 2.

Embodiment 77. The method of any of the preceding embodiments, wherein the dnase has at least 90% amino acid sequence identity with SEQ ID No. 2.

Embodiment 78. The method of any one of the preceding embodiments, wherein the dnase has at least 95% amino acid sequence identity with SEQ ID No. 2.

Embodiment 79. The method of any one of the preceding embodiments, wherein the dnase has at least 96% amino acid sequence identity with SEQ ID No. 2.

Embodiment 80. The method of any one of the preceding embodiments, wherein the dnase has at least 97% amino acid sequence identity with SEQ ID No. 2.

Embodiment 81. The method of any of the preceding embodiments, wherein the dnase has at least 98% amino acid sequence identity with SEQ ID No. 2.

Embodiment 82. The method of any of the preceding embodiments, wherein the dnase has at least 99% amino acid sequence identity with SEQ ID No. 2.

Embodiment 83 the method of any one of the preceding embodiments, wherein the dnase has 1,2,3, 4 or 5 substitutions, deletions or insertions compared to the amino acid sequence set forth in SEQ ID No. 2; preferably 1,2,3, 4 or 5 conservative substitutions.

Embodiment 84 the method of any one of the preceding embodiments, wherein the dnase is a NUC3 nuclease.

The method of any one of the preceding embodiments, wherein the dnase comprises the dnase_nuca_ NucB domain and further comprises any one of the motifs [ LV ] [ PTA ] [ FY ] [ DE ] [ VAGPH ] D [ CFY ] [ WY ] [ AT ] [ IM ] L [ CYQ ] and/or the motif GPYCK (SEQ ID NO: 3) or WF [ QE ] IT (SEQ ID NO: 4).

Embodiment 86. The method of any of the preceding embodiments, wherein the DNase has an amino acid sequence as shown in SEQ ID NO. 2.

Embodiment 87. The method of any of the preceding embodiments, wherein the microbial host cell is a bacterial host cell, a fungal host cell, or a yeast host cell.

Embodiment 88 the method of any one of the preceding embodiments, wherein the microbial host cell is a strain selected from the group consisting of: candida, hansenula, kluyveromyces, pichia, saccharomyces, schizosaccharomyces, yarrowia, acremonium, aspergillus, fusarium, humicola, mucor, myceliophthora, neurospora, penicillium, fusheca, torticola, and trichoderma.

Embodiment 89 the method of any of the preceding embodiments, wherein the microbial host cell is a strain of pichia.

Embodiment 90 the method of any one of the preceding embodiments, wherein the microbial host cell is a strain of pichia pastoris.

Embodiment 91. The method of any of the preceding embodiments, wherein the microbial host cell is a bacterial host cell.

Embodiment 92. The method of any of the preceding embodiments, wherein the microbial host cell is a strain selected from the group consisting of: bacillus, streptomyces, escherichia, butu and pseudomonas.

Embodiment 93 the method of any one of the preceding embodiments, wherein the microbial host cell is a strain of bacillus or escherichia.

Embodiment 94 the method of any one of the preceding embodiments, wherein the microbial host cell is a bacillus host cell; preferred are Bacillus amyloliquefaciens, bacillus licheniformis, or Bacillus subtilis host cells.

Embodiment 95. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 5ng/g recombinant DNA.

Embodiment 96. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 1ng/g recombinant DNA.

Embodiment 97. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.5ng/g recombinant DNA.

Embodiment 98. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.1ng/g recombinant DNA.

Embodiment 99. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.05ng/g recombinant DNA.

Embodiment 100. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.01ng/g recombinant DNA.

Embodiment 101. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises dnase.

Example 102A method for reducing the amount of DNA in a microbial fermentation product, the method comprising

(A) Providing a fermentation broth comprising bacillus host cells, recombinant DNA from the bacillus host cells, and enzymes produced by the bacillus host cells;

Embodiment 103. The method of embodiment 102, wherein the DNase is added to the fermentation medium before or during fermentation.

Embodiment 104. The method of embodiment 102, wherein the dnase is added to the fermentation broth in step (a).

Embodiment 105. The method of embodiment 102, wherein the dnase is added to the fermentation broth after step (a).

Embodiment 106. The method of embodiment 102, wherein the dnase is added to the fermentation broth after step (b).

Embodiment 107. The method of embodiment 102, wherein the dnase is added to the fermentation broth after step (c).

Embodiment 108. The method of any one of the preceding embodiments, wherein the enzyme is heterologous to the bacillus host cells.

Embodiment 109. The method of any of the preceding embodiments, wherein the enzyme is secreted into the fermentation broth by the bacillus host cells.

Embodiment 110. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises an enzyme in an amount of at least 0.1% w/w.

Embodiment 111. The method of any of the preceding embodiments, wherein the fermentation broth in step (a) comprises an enzyme in an amount of at least 0.5% w/w.

Embodiment 112. The method of any one of the preceding embodiments, wherein the fermentation broth in step (a) comprises an enzyme in an amount of at least 1% w/w.

Embodiment 113. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 50 kDa.

Embodiment 114. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 40 kDa.

Embodiment 115. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 30 kDa.

Embodiment 116. The method of the previous embodiment, wherein the membrane has a size exclusion limit of less than 20 kDa.

Embodiment 117. The method of the preceding embodiment, wherein the membrane has a size exclusion limit of less than 10 kDa.

Embodiment 118. The method of any one of the preceding embodiments, wherein the DNase has at least 85% amino acid sequence identity to SEQ ID NO. 2.

Embodiment 119. The method of any one of the preceding embodiments, wherein the dnase has at least 90% amino acid sequence identity with SEQ ID No. 2.

Embodiment 120. The method of any of the preceding embodiments, wherein the dnase has at least 95% amino acid sequence identity with SEQ ID No. 2.

Embodiment 121. The method of any of the preceding embodiments, wherein the dnase has at least 96% amino acid sequence identity with SEQ ID No. 2.

Embodiment 122. The method of any of the preceding embodiments, wherein the dnase has at least 97% amino acid sequence identity with SEQ ID No. 2.

Embodiment 123. The method of any of the preceding embodiments, wherein the dnase has at least 98% amino acid sequence identity with SEQ ID No. 2.

Embodiment 124. The method of any of the preceding embodiments, wherein the dnase has at least 99% amino acid sequence identity with SEQ ID No. 2.

Embodiment 125 the method of any one of the preceding embodiments, wherein the dnase has 1,2,3,4 or 5 substitutions, deletions or insertions compared to the amino acid sequence set forth in SEQ ID No. 2; preferably 1,2,3,4 or 5 conservative substitutions.

Embodiment 126 the method of any one of the preceding embodiments, wherein the dnase is a NUC3 nuclease.

Embodiment 127. The method of any of the preceding embodiments, wherein the dnase comprises the dnase_nuca_ NucB domain and further comprises any of the motifs [ LV ] [ PTA ] [ FY ] [ DE ] [ VAGPH ] D [ CFY ] [ WY ] [ AT ] [ IM ] L [ CYQ ] and/or the motif GPYCK (SEQ ID NO: 3) or WF [ QE ] IT (SEQ ID NO: 4).

Embodiment 128 the method of any one of the preceding embodiments, wherein the dnase has an amino acid sequence as set forth in SEQ ID No. 2.

Embodiment 129 the method of any of the preceding embodiments, wherein the bacillus host cell is a bacillus amyloliquefaciens, bacillus licheniformis, or bacillus subtilis host cell.

Embodiment 130. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 5ng/g recombinant DNA.

Embodiment 131. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 1ng/g recombinant DNA.

Embodiment 132 the method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.5ng/g recombinant DNA.

Embodiment 133. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.1ng/g recombinant DNA.

Embodiment 134. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.05ng/g recombinant DNA.

Embodiment 135. The method of any of the preceding embodiments, wherein the microbial fermentation product comprises less than 0.01ng/g recombinant DNA.

Embodiment 136. The method of any one of the preceding embodiments, wherein the microbial fermentation product comprises a dnase.

Example 137A microbial fermentation product comprising less than 10ng/g recombinant DNA.

Example 138 the microbial fermentation product of the previous example comprising less than 5ng/g recombinant DNA.

Embodiment 139. The microbial fermentation product of any one of the preceding embodiments, comprising less than 1ng/g recombinant DNA.

Embodiment 140 the microbial fermentation product of any one of the preceding embodiments, comprising less than 0.5ng/g recombinant DNA.

Embodiment 141. The microbial fermentation product of any one of the preceding embodiments, comprising less than 0.1ng/g recombinant DNA.

Embodiment 142. The microbial fermentation product of any one of the preceding embodiments, comprising less than 0.05ng/g recombinant DNA.

Example 143 the microbial fermentation product of any one of the preceding examples, comprising less than 0.01ng/g recombinant DNA.

Embodiment 144 the microbial fermentation product of any one of the preceding embodiments, further comprising a fungal dnase or variant thereof.

Embodiment 145. The microbial fermentation product of the previous embodiment, wherein the fungal dnase or variant thereof is a NUC3 nuclease.

Embodiment 146 the microbial fermentation product of any one of the preceding embodiments, comprising a protein of interest.

Example 147. The microbial fermentation product of the previous example, wherein the protein of interest is a recombinant protein of interest.

Embodiment 148 the microbial fermentation product of any of the preceding embodiments comprising at least 0.1% w/w protein of interest.

Embodiment 149. The microbial fermentation product of any one of the preceding embodiments, comprising at least 0.5% w/w of the protein of interest.

Embodiment 150. The microbial fermentation product of any one of the preceding embodiments, comprising at least 1% w/w protein of interest.

Embodiment 151. The microbial fermentation product of the previous embodiment, wherein the protein of interest is an enzyme.

Embodiment 152 the microbial fermentation product of any one of the preceding embodiments produced by the method of any one of the preceding embodiments.

Examples

Chemicals are at least reagent grade commodity products.

DNAzymes

A "bacterial nuclease" is a Bacillus subtilis DNase having an amino acid sequence as shown in SEQ ID NO. 1.

A "fungal nuclease" is an Aspergillus oryzae DNase having an amino acid sequence as shown in SEQ ID NO. 2.

Other dnases were also used for comparison. Donor strains are shown in the corresponding examples.

The detection of gDNA and resDNA was performed by PCR and digital PCR (dPCR) analysis described in example 1. The specific analysis used will be described in the separate examples.

Example 1

Detection of residual recombinant DNA

The method for proving the absence of residual recombinant DNA in each sample can be divided into 2 steps: step i) extracting DNA from the sample, including a lysis step, and step ii) amplifying by PCR using conventional PCR techniques followed by gel electrophoresis or digital PCR techniques to detect recombinant DNA (target).

DNA extraction comprising a cleavage step

The DNA extraction method is based on commercial kits (Maxwell RSC PureFood GMO and certified kits (Maxwell RSC PureFood GMO & Authentication kit), promega). The standard treatment with proteinase K was modified to allow efficient removal of protein, thus reducing the likelihood of PCR inhibition by the sample matrix.

First, 200. Mu.l of the sample was treated with proteinase K (QIAgen catalog number 19133) in CTAB buffer to a final concentration of 12.5mg proteinase K/ml sample. The mixture was then incubated at 40 ℃ for 1 hour to ensure efficient removal of the protein. Subsequently, DNA was extracted using a Maxwell RSC instrument following the procedure described in the commercial kit, however, DNA was eluted in 55 μl dnase free water.

Detection of residual recombinant DNA using PCR amplification

PCR amplification is a common method for detecting very small amounts of target DNA. In these experiments, both conventional PCR and digital PCR techniques were used. The difference between these two PCR methods is as follows: traditional PCR techniques are a qualitative approach in which amplified PCR targets are detected by agarose gel electrophoresis. However, the digital PCR technique is a quantitative method based on the TaqMan technique, in which the amount of amplified PCR target can be quantified.

Primers for both techniques are unique to the recombinant DNA in question (table 1) and will amplify target fragments less than 150bp in size. For digital PCR techniques based on TaqMan techniques, sequence-specific oligonucleotides (probes) with fluorophore and quencher moieties attached are also required (table 1). The design of primers, probes and amplicons is based on the basic considerations of use webtools, as described by Rodriguez et al at Chapter 3:Design of primers and Probes for Quantitative Real-time PCR methods in Methods in Molecular Biology 1275[ chapter 3: primer and probe set-up for quantitative real-time PCR method in molecular biology method 1275, springer Protocols [ Springer laboratory Manual ] (edit C.Basu).

TABLE 1 PCR primers used for conventional PCR amplification.

TABLE 2 PCR primers and probes for digital PCR amplification.

TABLE 3 PCR primers used for conventional PCR amplification.

TABLE 4 PCR primers used for conventional PCR amplification.

TABLE 5 PCR primers and probes for digital PCR amplification.

TABLE 6 PCR primers and probes for digital PCR amplification.

Traditional PCR method

10. Mu.L of extracted DNA was PCR amplified using the relevant primer set for the given target at a concentration of 400nM for each primer. Each reaction used a tube of Illustra PureFood PCR bead system (catalog number 27-9557-02) from GE healthcare group (GE HEALTHCARE). The PCR reaction was performed under the following thermocycler conditions:

After PCR amplification, the PCR reaction was visualized on a 2.2% agarose gel (FlashGel systems from Lonza, cat. No. 57031). FLASHGEL DNA tag 100bp-4kb (Dragon company catalog number 50473) was used for band size estimation.

Digital PCR method

Digital PCR (dPCR) uses the procedure of end-point PCR, but divides the PCR reaction into a number of single partitions, with templates randomly distributed across all available partitions. After PCR, amplification was detected by measuring fluorescence in all positive partitions. Using poisson statistics, the average amount of target DNA per sample can be calculated. The number of filled partitions is identified by the reference fluorescent dye present in the reaction mixture. An absolute quantification of the amount of target DNA in each sample can then be calculated.

For these experiments, a digital system QIAcuity ONE from the company qiagen was used with the QIAcuity probe PCR kit (catalog number 250101). The kit contains a 4x concentrated ready-to-use master mix optimized for use with QIAcuity nm plates (catalog number 25001) that partition each sample into 26,000 partitions. The procedure described by the supplier has been optimized in terms of primer and probe concentrations and PCR cycling conditions. For these experiments, a primer concentration of 800nM and a probe concentration of 400nM each were used.

The extracted DNA should be fragmented by restriction enzyme cleavage prior to partitioning to ensure uniform distribution of the template throughout the QIAcuity nm plate. For these experiments, the restriction enzyme EcoRI was selected at a concentration of 0.25U/40. Mu.l of reaction. Such restriction enzymes are chosen because they do not cleave within the target fragment. For each dPCR reaction, 5 μl of template DNA was used in a total reaction volume of 40 μl obtained using dnase-free water.

The mixture was then transferred to the nanoplates, sealed, and incubated at room temperature for at least 10 minutes to allow the restriction enzyme to function.

Immediately thereafter, the nanoplates were run under the following thermal cycling conditions:

The result of the dPCR analysis is given as copy number of target PCR fragment per μl total reaction volume. Based on this value, the amount of ng recombinant DNA per g sample in the starting material can be calculated based on the following assumption:

100% efficient DNA purification was obtained.

The average weight of the Bacillus genome is estimated to be 4.2X10 ⁶ base pairs (cf. J.T.Trevors,1996,Genome size in bacteria [ bacterial genome size ]. Antonie van Leeuwenhocek 69 (4): 293-303).

The molecular weight of the base pairs is 650 daltons.

One daltons corresponds to 1.6605x10 ^-15 ng.

The weight of the Bacillus genome was estimated to be 4.5X10 ^-6 ng.

The content of residual recombinant DNA in the initial sample can then be calculated using the following equation:

ng DNA/ml sample = 0.04x Df x D

-Wherein Df is the dilution factor of the DNA preparation before dPCR analysis and D is the result of dPCR analysis expressed as copy number per μl in 40 μl total reaction volume.

Example 2

Degradation of genomic DNA in distilled water by bacterial nucleases from food bacillus and fungal nucleases from Aspergillus oryzae

Experimental procedure

To understand nuclease degradation of purified bacillus licheniformis genomic DNA (gDNA) in a simple "clean" matrix, all samples in example 2 were prepared in Distilled Water (DW). To evaluate gDNA degradation, two positive control samples were prepared in DW, wherein the total volume of each sample was 300 μl. Control samples were spiked with 1000ng/g gDNA at pH 9. To one of the two control samples, a total of 5mM MgCl ₂ was added. Another positive control sample was prepared in DW and incorporated with 10ng/g gDNA. Dose response studies were performed on degradation of gDNA using bacterial nucleases with final concentrations ranging from 0.0005g/L to 0.5g/L with a concentration increment factor of 10. In addition, mgCl ₂ (cofactor) was added to an additional sample at a final concentration of 0.005g/L to a final concentration of 5mM. A final concentration of 0.5g/L was used for the fungal nuclease. The total reaction volume was 300 μl at pH 9, and the samples were incubated at 37 ℃ with gentle agitation for 24 hours. The nuclease reaction was quenched by freezing the sample after the incubation time. The ability of nucleases to degrade gDNA was assessed by conventional PCR analysis using the primers shown in table 4.

Results

Both bacterial and fungal nucleases were able to degrade gDNA to <10ng/g in DW at any concentration tested, and independent of whether additional MgCl ₂ was added (table 7).

Table 7.

Example 3

Degradation of DNA in enzyme concentrates by bacterial and fungal nucleases

Experimental procedure

All samples in example 3 were prepared using an enzyme concentrate recovered from bacillus licheniformis broth (FB). Residual DNA (resDNA) concentration of enzyme concentrate >10ng/g. Residual DNA is the amount of recombinant DNA derived from the bacillus licheniformis host cell. Dose response studies were performed on ResDNA degradations using bacterial nucleases, with final concentrations ranging from 0.0005g/L to 0.5g/L, with a concentration increment factor of 10. In addition, mgCl ₂ (cofactor) was added to an additional sample at a final concentration of 0.005g/L to a final concentration of 5mM. A final concentration of 0.5g/L was used for the fungal nuclease. The total reaction volume was 400 μl at pH 6.5, and the samples were incubated at 37 ℃ with gentle agitation for 24 hours. The nuclease reaction was quenched by freezing the sample after the incubation time. Two positive control samples were used: an enzyme concentrate and DW were incorporated at 10ng/g gDNA. resDNA degradation was assessed by conventional PCR analysis using the primers shown in table 3.

Results

The fungal nuclease is capable of degrading the bacterial host cells resDNA in the enzyme concentrate to < <10ng/g. Bacterial nucleases were unable to degrade any significant level of bacterial host cells resDNA at any of the concentrations tested, and were independent of whether additional MgCl ₂ was added (table 8).

Table 8.

Example 4

Degradation of DNA in enzyme concentrates by bacterial and fungal nucleases at different pH

Experimental procedure

Since the fungal nuclease is the only nuclease that effectively degrades resDNA in example 3, the test was repeated at different pH in the enzyme concentrate recovered from B.licheniformis FB. The resDNA concentration of the enzyme concentrate was >10ng/g. A final concentration of 0.5g/L was used for both nucleases. The total reaction volume was 400 μl at pH 4,5, 6, 7 and 8 (fungal nuclease) and pH 4,6 and 8 (bacterial nuclease), and the samples were incubated with gentle agitation for 24 hours at 37 ℃. The nuclease reaction was quenched by freezing the sample after the incubation time. An enzyme concentrate without any nuclease added was used as a positive control sample. resDNA degradation was assessed by conventional PCR analysis using the primers shown in table 1.

Results

At all pH values studied, fungal nucleases were able to degrade bacterial host cells resDNA in the enzyme concentrate to < <10ng/g, but most efficient at pH 4 (< <10 ng/g). At any of the tested pH, the bacterial nucleases were unable to degrade any significant level of bacterial host cells resDNA in the enzyme concentrate, but a slight effect was observed at pH 4 (table 9).

Table 9.

Sample type	ResDNA concentration
		Control sample-enzyme concentrate	>10ng/g
Fungal nuclease-pH 4	<<<10ng/g
		Fungal nuclease-pH 5	<<10ng/g
Fungal nuclease-pH 6	<<10ng/g
		Fungal nuclease-pH 7	<<10ng/g
Fungal nuclease-pH 8	<<10ng/g
		Bacterial nuclease-pH 4	<10ng/g
Bacterial nuclease-pH 6	≥10ng/g
		Bacterial nuclease-pH 8	≥10ng/g

Example 5

Degradation of DNA in fermentation and flocculation broths by fungal nucleases at different pH conditions

Experimental part

All samples in example 5 were prepared using FB or flocculated fermentation broth (fFB) from bacillus licheniformis.

Preparation of fermentation broth samples

Fungal nuclease was added directly to FB at pH 7.5+/-0.5 to reach a final nuclease concentration of 0.5 g/L. FB without any fungal nuclease added was used as a positive control sample. The samples were incubated at 37℃for 24 hours with gentle agitation. The nuclease reaction was quenched by freezing the sample after the incubation time. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 2.

Preparation of fFB samples

To generate fFB, FB was processed as follows:

1. Diluting with water

2. Addition of divalent salts

3. Addition of polyaluminum chloride (PAC)

4. Adjusting pH to 4, 6 or 8

5. Adding fungal nuclease to achieve a final concentration of 0.5g/L

6. Incubation was carried out at 37℃for 24 hours with gentle agitation

Positive control samples were prepared in the same manner, but step 5 was not performed. The nuclease reaction was quenched by freezing the sample after the incubation time. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 2.

Results

The fungal nuclease significantly degrades the bacterial host cell resDNA, whether it is added to FB or fFB, compared to the control sample. Furthermore, the efficiency of the nuclease was similar at all tested pH (table 10).

Table 10.

Example 6

Degradation of DNA during recovery of enzymes from fermentation broths

Experimental part

During recovery of the enzyme from bacillus licheniformis FB, all samples in example 6 were from different streams.

Preparation of FB

Fungal nuclease was added to the tank before the fermentation began to reach a final nuclease concentration of 0.5g/L +/-0.1. The pH during fermentation and recovery was 7.5+/-0.5. FB without any fungal nuclease added was used as a positive control batch. FB was harvested and incubated at 5 ℃ for 22-24 hours before recovery was started. The nuclease reaction was quenched by freezing the sample after the incubation time. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 2.

Recovery process

To generate fFB, FB was processed as follows:

1. Diluting with water

2. Addition of divalent salts

3. Addition of polyaluminum chloride (PAC)

4. The pH was adjusted to 7.5+/-0.5

5. Addition of cationic Polymer

6. Addition of anionic Polymer

7. FFB is centrifuged to separate the liquid phase (supernatant) from the biomass

8. Filtering the supernatant on a filter with a cut-off size of 5.0-0.3 μm to produce a filtrate

9. Ultrafiltration (UF) concentrate filtrate using membrane cut-off size of 10kDa to produce UF concentrate

Results

Fungal nucleases significantly degraded bacterial host cells resDNA in FB, FB supernatant and UF concentrate compared to control FB (table 11).

Table 11.

Example 7

Degradation of DNA in enzyme concentrates using DNase from different organisms

Experimental procedure

All samples in example 7 were prepared using an enzyme concentrate recovered from B.licheniformis FB at resDNA concentration >10ng/g. The use of resDNA degradation of nucleases from different organisms was investigated at a final nuclease concentration of 0.5 g/L. The enzyme concentrate had a pH of 5.9 and the sample was incubated at 25 ℃ for 1 hour with gentle agitation. The nuclease reaction was quenched by freezing the sample after the incubation time. The enzyme concentrate was used as a positive control sample. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 5.

Results

Fungal nucleases (from aspergillus oryzae) degraded bacterial host cells resDNA in the enzyme concentrate to < <1ng/g, with no other test nucleases from different organisms degrading resDNA to <10ng/g (table 12).

Table 12.

Example 8

Degradation of DNA in enzyme concentrates using DNase from different organisms

Experimental procedure

All samples in example 8 were prepared using an enzyme concentrate recovered from bacillus licheniformis FB at resDNA concentration >10ng/g. The use of resDNA degradation of nucleases from different organisms was investigated at a final nuclease concentration of 0.5 g/L. The enzyme concentrate had a pH of 5.0 and the sample was incubated at 25 ℃ for 1 hour with gentle agitation. The nuclease reaction was quenched by freezing the sample after the incubation time. The enzyme concentrate was used as a positive control sample. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 2.

Results

Fungal nucleases (from aspergillus oryzae) degrade bacterial host cells resDNA in the enzyme concentrate to < <1ng/g. Bacterial nucleases and nucleases from Ma Saxin sartolla, arthrobacter species 07MA20, rhizoctonia solani and Morchella gravida degrade resDNA to <10ng/g. Nucleases from aschersonia species, aschersonia aleyrodis, penicillium quercetin, curvularia gondii and cephalosporanges were unable to degrade resDNA <10ng/g (table 13).

Table 13.

Example 9

Degradation of DNA in enzyme concentrates using DNase from different organisms

Experimental procedure

All samples in example 9 were prepared using an enzyme concentrate recovered from bacillus subtilis FB, resDNA concentration >10ng/g. The use of resDNA degradation of nucleases from different organisms was investigated at a final nuclease concentration of 0.5 g/L. The enzyme concentrate had a pH of 6.2 and the sample was incubated at 25 ℃ for 1 hour with gentle agitation. The nuclease reaction was quenched by freezing the sample after the incubation time. The enzyme concentrate was used as a positive control sample. resDNA degradation was assessed by dPCR analysis using the primers/probes shown in table 6.

Results

Fungal nucleases (from aspergillus oryzae) degrade bacterial host cells resDNA in the enzyme concentrate to < <1ng/g. Bacterial nucleases and nucleases from Ma Saxin sarcina, echinococcus species, dactylotheca reesei and curvularia gondii were unable to degrade resDNA to <10ng/g (table 14).

Table 14.

Sample type	ResDNA concentration
		Control sample-enzyme concentrate	>>>10ng/g
Fungal nucleases (from Aspergillus oryzae)	<<1ng/g
		Bacterial nucleases (from food bacillus)	>>10ng/g
DNase from Ma Saxin sartolla	>>10ng/g
		DNase from Aminococcus species	>>10ng/g
DNase from Rhizopus arundinaceus	>>10ng/g
		DNase from Curvularia gondii	>>10ng/g

Claims

1. A method for reducing the amount of DNA in a microbial fermentation product, the method comprising

2. The method of the preceding claim, wherein the protein of interest is heterologous to the microbial host cells.

3. The method of any one of the preceding claims, wherein the fermentation broth in step (a) comprises at least 0.1% w/w; preferably at least 0.5% w/w; more preferably at least 1% w/w of the protein of interest.

4. The method of any one of the preceding claims, wherein the protein of interest is an enzyme, heme-containing protein, cell signaling protein or ligand binding protein, preferably an enzyme.

5. The method of any one of the preceding claims, wherein the membrane filtration in step (c) comprises a microfiltration step.

6. The method of any one of the preceding claims, wherein the membrane filtration in step (c) comprises an ultrafiltration step.

7. The method of any one of the preceding claims, wherein some or all of the microbial host cells in step (a) have been mechanically or enzymatically disrupted/homogenized.

8. The method of any one of the preceding claims, wherein the fungal dnase or variant thereof has at least 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or 100% amino acid sequence identity with SEQ ID No. 2, and wherein the variant exhibits dnase activity.

9. The method of any one of the preceding claims, wherein the fungal dnase or variant thereof comprises the dnase_nuca_ NucB domain and further comprises any one of the motifs [ LV ] [ PTA ] [ FY ] [ DE ] [ VAGPH ] D [ CFY ] [ WY ] [ AT ] [ IM ] L [ CYQ ] and/or motif GPYCK (SEQ ID NO: 3) or WF [ QE ] IT (SEQ ID NO: 4).

10. The method of any one of the preceding claims, wherein the microbial host cell is a strain selected from the group consisting of: candida, hansenula, kluyveromyces, pichia, saccharomyces, schizosaccharomyces, yarrowia, acremonium, aspergillus, fusarium, humicola, mucor, myceliophthora, neurospora, penicillium, fusheca, torticola, and trichoderma.

11. The method of any one of the preceding claims, wherein the microbial host cell is a bacterial host cell; preferably selected from the group consisting of: bacillus, streptomyces, escherichia, branchia and pseudomonas; more preferably a strain of bacillus.

12. The method of any one of the preceding claims, wherein the microbial fermentation product comprises less than 10ng/g recombinant DNA; preferably less than 5ng/g, less than 1ng/g, less than 0.5ng/g, less than 0.1ng/g, less than 0.05ng/g or less than 0.01ng/g of recombinant DNA.

13. A microbial fermentation product comprising less than 10ng/g recombinant DNA; preferably less than 5ng/g, less than 1ng/g, less than 0.5ng/g, less than 0.1ng/g, less than 0.05ng/g or less than 0.01ng/g of recombinant DNA.

14. The microbial fermentation product of claim 13, further comprising a fungal dnase or variant thereof, preferably a NUC3 nuclease.

15. The microbial fermentation product of claim 13 or 14, further comprising a protein of interest; preferably at least 0.1% w/w of the protein of interest; more preferably at least 0.5% w/w of the protein of interest; even more preferably at least 1% w/w of the protein of interest.

16. The microbial fermentation product of claim 15, wherein the protein of interest is a recombinant protein of interest; enzymes are preferred.

17. The microbial fermentation product of any one of claims 13-16 produced by the method of any one of the preceding claims.