WO2013119340A1

WO2013119340A1 - Microorganism and process for isoprene production

Info

Publication number: WO2013119340A1
Application number: PCT/US2013/020651
Authority: WO
Inventors: Stephanie DONESKE; Paul Campbell
Original assignee: Glycos Biotechnologies, Inc.
Priority date: 2012-02-08
Filing date: 2013-01-08
Publication date: 2013-08-15

Abstract

The present invention provides non-naturally occurring microorganisms that have been modified to produce isoprene. The microorganisms include a 3-hydroxy-3- methylglutaryl-CoA reductase that uses NADPH as a co-factor, and overexpression of a pyridine nucleotide transhydrogenase. Further embodiments provide for methods to produce isoprene using non-naturally occurring microorganisms.

Description

MICROORGANISM AND PROCESS FOR ISOPRENE PRODUCTION

RELATED APPLICATION

[001] This application claims the benefit of U.S. Provisional Application No.

61/633,267, filed on February 8, 2012. The entire teaching of the above application is incorporated herein by reference.

FIELD OF THE INVENTION

[002] The present disclosure generally relates to the use of a non-naturally occurring microorganism for the production of isoprene. More specifically, the present disclosure relates to non-naturally occurring microorganisms that have been modified to overexpress the genes pntA and pntB to modify the NADH/NADPH balance of the microorganism to improve the production of isoprene.

BACKGROUND OF THE INVENTION

[003] Currently, many high-value chemicals or fuels are typically manufactured by chemical synthesis from hydrocarbons, including petroleum oil and natural gas. Also, high value chemicals may be produced as "by-products" during the processing of crude oil into usable fractions. For example, isoprene has typically been produced during the catalytic cracking of crude oil. However, as catalytic crackers have shifted their focus from crude oil to natural gas, there is now a reduced source of the four and five carbon chain molecules that are found in crude oil, but not natural gas.

[004] Being a short-chain carbon source, isoprene is a useful early or initial component for synthesizing a variety of chemicals. Isoprene may be used as a monomer or co-monomer. Examples of chemicals that can be produced using isoprene include polyisoprene, polybutylene, styrene-isoprene-styrene block co-polymers, and others. An example of an industry that uses isoprene is the synthetic rubber industry.

[005] Given the demand for and the many uses of isoprene, a new method of isoprene production is desired. Also, as the concerns of energy security, increasing oil and natural gas prices, and global warming escalate, the chemical production industry is seeking ways to replace chemicals made from non-renewable feedstocks with chemicals produced from renewable feedstocks using environmentally friendly practices.

[006] Thus, there is a need and a desire for a biological method of isoprene production. SUMMARY OF THE INVENTION

[007] Embodiments of the present invention generally provide non-naturally occurring microorganisms and methods to produce isoprene by fermentation. In one preferred embodiment, the non-naturally occurring microorganisms express a heterologous isoprene pathway in sufficient quantities to convert acetyl-CoA into isoprene. The isoprene pathway is described as heterologous because one or more genes encoding activities in the pathway are encoded by or derived from one or more different microorganisms than the non-naturally occurring microorganism. The isoprene pathway includes a 3-hydroxy-3-methylglutaryl- CoA reductase that uses NADPH as a co-factor. In addition to the 3 -hydroxy - 3methylglutaryl-CoA reductase, which may also have thiolase activity, the isoprene pathway typically further comprises a 3-hydroxy-3-methylglutaryl-CoA synthase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, an isopentenyl diphosphate isomerase, and an isoprene synthase. The microorganisms also preferably overexpress a pyridine nucleotide transhydrogenase activity, e.g., at levels higher than those produced by the parental strain of the microorganisms. The overexpression may be achieved by different means, such as by introducing multiple copies of the pyridine nucleotide transhydrogenase into the microorganisms, and/or overexpressing an endogenous (or heterologous) pyridine nucleotide transhydrogenase, such as with a strong promoter.

[008] An additional preferred embodiment provides a method of producing isoprene involving culturing non-naturally occurring microorganisms that express a heterologous 3- hydroxy-3-methylglutaryl-CoA reductase that uses NADPH as a co-factor, and overexpresses a pyridine nucleotide transhydrogenase activity in a suitable medium for the production of isoprene and collecting the isoprene that is produced.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

[010] Figure 1 shows a biosynthetic isoprene pathway.

[01 1] Figure 2 shows the pyridine nucleotide transhydrogenase activity. [012] Figure 3 shows the DNA sequence of plasmid pGA31R-MCS (SEQ ID NO.: 1).

[013] Figure 4 shows the DNA sequence of plasmid pGE21R-MCS (SEQ ID NO.: 2).

[014] Figure 5 shows the codon-optimized sequence of the mvaE and mvaS genes of Enterococcus faecalis ATCC 700802 (SEQ ID NO.: 3), including incorporated ribosomal binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

[015] Figure 6 shows the codon-optimized sequence (SEQ ID NO.: 4) of the synthetic operon encoding the mevalonate kinase gene of Methanocaldococcus jannaschi, the phosphomevalonate kinase of Enterococcus faecalis ATCC 700802, the mevalonate pyrophosphate decarboxylase of Saccharomyces cerevisiae S288C, and the isopentenyl diphosphate isomerase gene of E. coli MG1655, including incorporated ribosomal binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

[016] Figure 7 shows the codon-optimized sequence of the synthetic isoprene synthase gene ofPopulus alba (SEQ ID NO.: 5), containing a ribosomal binding site and flanking restriction endonuclease sites used in subsequent cloning steps, but without the encoded N- terminal transit peptide.

[017] Figure 8 shows a cloning strategy for the production of plasmids pGB 1004, pGB1012 and pGB1017.

[018] Figure 9 shows a cloning strategy for the production of plasmids pGB 1008 and pGB1026.

[01 ] Figure 10 shows a cloning strategy for the production of plasmids pGB 1030 and pGB1033.

[020] Figure 11 shows a comparison of MG1655 harboring plasmids pGB1012 and pGB1030 ("no pntAB") and MG1655 harboring plasmids pGB 1012 and pGB1033 ("with pntAB").

[021 ] Figure 12 shows a comparison of FA01 harboring plasmids pGB 1017 and pGB1030 ("no pntAB") and FA01 harboring plasmids pGB 1017 and pGB1033 ("with pntAB").

DETAILED DESCRIPTION

[022] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word "a" or "an" means "at least one", and the use of "or" means "and/or", unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.

[023] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[024] As defined herein, a knocked-out gene is a gene whose encoded product, e.g., a protein, does not or substantially does not perform its usual function or any function. A knocked-out gene can be created through deletion, disruption, insertion, or mutation. As defined herein, microorganisms that lack one or more indicated knocked-out genes are also considered to have knock outs of the indicated gene(s). The microorganisms themselves may also be referred to as knock outs of the indicated gene(s). Such knock outs can also be conditional or inducible, using techniques that are well-known to those of skill in the art. Also contemplated are "knock ins", in which a gene, or one or more segments of a gene, are introduced into the microorganism in place of, or in addition to, the endogenous copy of the gene. Once again, many techniques for creating knock in microorganisms are known to those of ordinary skill in the art.

[025] A non-naturally occurring microorganism is defined herein to include a microorganism that has been modified by recombinant DNA or other techniques. Preferably, the microorganisms are bacteria and contain one or more heterologous genes or segments of genes that express one or more heterologous proteins or activities.

[026] The methods and techniques utilized for culturing or generating the

microorganisms disclosed herein are known to the skilled worker trained in microbiological and recombinant DNA techniques. Methods and techniques for growing microorganisms (e.g., bacterial cells), transporting isolated DNA molecules into the host cell and isolating, cloning and sequencing isolated nucleic acid molecules, knocking out expression of specific genes, etc., are examples of such techniques and methods. These methods are described in many items of the standard literature, which are incorporated herein in their entirety: "Basic Methods In Molecular Biology" (Davis, et ah, eds. McGraw-Hill Professional, Columbus, OH, 1986); Miller, "Experiments in Molecular Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1972); Miller, "A Short Course in Bacterial Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992); Singer and Berg, "Genes and Genomes" (University Science Books, Mill Valley, CA, 1991); "Molecular Cloning: A Laboratory Manual," 2^nd Ed. (Sambrook, et ah, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); "Handbook of Molecular and Cellular Methods in Biology and Medicine" (Kaufman, et al, eds., CRC Press, Boca Raton, FL, 1995); "Methods in Plant Molecular Biology and Biotechnology" (Glick and Thompson, eds., CRC Press, Boca Raton, FL, 1993); and Smith-Keary, "Molecular Genetics of Escherichia coi (The Guilford Press, New York, NY, 1989).

[027] The conversion of acetyl coenzyme A (acetyl-CoA) to isoprene occurs through a series of eight steps (Figure 1). The third step in the process, the conversion of 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA) to mevalonic acid (MVA, also referred to as mevalonate) requires two reducing equivalents in the form of NADH or NADPH. While 3-hydroxy-3- methylglutaryl-CoA reductases that use NADH as a cofactor are known, many members of this family of enzymes use NADPH in place of or in addition to NADH. In contrast, the primary metabolic pathways to produce acetyl-CoA generate NADH, rather than NADPH, as a cofactor.

[028] Most microorganisms use glycolytic pathways to produce acetyl-CoA from carbon sources such as glucose or glycerol. For example, E. coli uses the Embden Meyerhof pathway to convert glucose to acetyl-CoA. One key glycolytic step, the conversion of D- glyceraldehyde-3 -phosphate to 1,3-bisphospho-D-glycerate, generates NADH, not NADPH. Likewise, under aerobic conditions, the pyruvate dehydrogenase complex converts pyruvate, derived from glucose or glycerol, to acetyl-CoA with the generation of NADH.

[029] The production of isoprene and related molecules from acetyl-CoA via 3 -hydroxy - 3-methylglutaryl-CoA reductase may be limited by the availability of NADPH to use as a cofactor by HMG-CoA reductase. One method of alleviating the limiting levels of NADPH is to shift the pool of reducing equivalents from NADH to NADPH using a pyridine nucleotide transhydrogenase. [030] The primary source of NADPH in E. coli is supplied through the conversion of NADH to NADPH by the pyridine nucleotide transhydrogenase, PntAB, encoded by the pntAB locus. This membrane-bound enzyme couples the inward movement of protons across the membrane with the reduction of NADP⁺ by NADH, yielding NADPH and NAD⁺. In E. coli, a second, soluble pyridine nucleotide transhydrogenase, the SthA enzyme encoded by sthA, is involved in the reoxidation of NADPH to NADP⁺ with the concomitant conversion of NAD⁺ to NADH; however, under some conditions such as the availability of excess NADH, SthA can carry out the reverse reaction.

[031] While the 3-hydroxy-3-methylglutaryl-CoA reductase used in the examples presented here is encoded by the codon-optimized mvaE gene of Enterococcus faecalis

ATCC 700802 as presented in Figure 5, it can be appreciated by one who is skilled in the art that other 3-hydroxy-3-methylglutaryl-CoA reductase enzymes that can use NADPH as a cofactor will benefit from the elevated levels of NADPH generated through the conversion of NADH to NADPH by, for example, transhydrogenase activities. Non-limiting examples of 3-hydroxy-3-methylglutaryl-CoA reductase enzymes that use NADPH exclusively or in addition to NADH are presented in Table 3. These include, but are not limited to, the activities encoded by the following genes: HMG1 of Arabidopsis thaliana; hmgA of

Haloferax volcanii; HMGR1 oiHevea brasiliensis; HMGCR of Homo sapiens; HMG1 of Saccharomyces cerevisiae; mvaA of Staphylococcus aureus; hmgr of Streptomyces sp. Strain CL190; and hmgA of Sulfolobus solfataricus. While membrane-bound pyridine nucleotide transhydrogenase activity encoded by pntAB ofE. coli, for example, or soluble pyridine nucleotide transhydrogenase activity encoded by sthA ofE. coli, for example, may be used to facilitate the conversion of NADH to NADPH, genes encoding other suitable pyridine nucleotide transhydrogenase activities may be used as well.

[032] Thus, an approach to improving isoprene production from acetyl-CoA via 3- hydroxy-3-methylglutaryl-CoA reductases that can use NADPH as a cofactor is to increase the availability of NADPH by overexpression of genes encoding pyridine nucleotide transhydrogenases. Although many such transhydrogenases are known in the literature, PntAB and SthA, as native E. coli genes, are ideal candidates for altering cofactor availability in E. co/z^'-based systems. STRAINS

The common E. coli cloning strains DHIOB and DH5a were used during construction of all vectors. For testing the plasmid constructs, MG1655 was used as the host strain. DNA acquired through complete synthesis was received already transformed in DHIOB. Vectors that were constructed in house were transformed into chemically competent DH5 a cells (GC5, Gene Choice, available from Sigma-Aldrich Co. LLC). Wild type MG1655 was obtained from the University of Wisconsin E. coli Genome Project

(https://www.genome.wisc.edu) (Kang et al, 2004). MG1655 was made electrocompetent and electroporated following the protocol from the MicroPulser Electroporation Apparatus Operating Instructions and Applications Guide (Bio-Rad catalog number 165-2100), except that LB without salt was used to grow up the culture in making cells electrocompetent. Strain genotypes are found in Table 1.

Table 1

STRAINS

Strain Genotype Reference

MG1655 Wild type E. coli (F^{" "} ilvCT rjb-50 rph-1) ATCC¹ # 47076

DH10B E. coli (F^" endAl recAl galE15 galK16 nupG rpsL DNA2.0, www.dna20.com

AlacX74 <D801acZAM15 araD139 A(ara,leu)7697 mcrA

A(mrr-hsdRMS-mcrBC) λ^"'

GC5 (DH5a) E. coli (F^"cp801acZAM15 A(lacZYA-argF)U169 recAl Sigma-Aldrich Co. LLC, www.sigmaaldrich.com endAl hsdR17(rk^", mk⁺) phoA supE44 thi-1 gyrA96 relAl

λ^" tonA)

FA01 E. coli MG1655 fadR* atoC" Dellomonaco, C, Rivera, R.,

Campbell, P., and R. Gonzalez. 2010. Engineered Respiro- Fermentative Metabolism for the Production of Biofuels and Biochemicals from Fatty Acid- Rich Feedstocks. Appl. Env. Microbiol. 76: 5067 - 5078. ' American Type Culture Collection

CLONING METHODS AND PLASMIDS

[033] Standard methods were used for construction of the plasmids and polymerase chain reaction (Miller, 1992; Sambrook and Russell, 2001). AccuPrime Pfx (Invitrogen) used when amplifying genomic DNA. Plasmid isolation was performed using Zyppy Plasmid Miniprep Kit (Zymo Research Corp), gel extraction was done using Zymoclean Gel DNA Recovery Kit (Zymo Research Corp), PCR purification was performed using

QuickClean 5M PCR Purification Kit (GenScript), and genomic DNA isolation performed using Gentra Puregene Yeast and Bacteria Kit (Qiagen). All restriction enzymes were obtained from NEB. Many components were synthesized by DNA2.0 or GenScript.

Plasmids used in this project are listed in Table 2. Enzymes and the associated gene names are as set forth in Table 3.

Table 2

PLASMIDS

Strain Features Source

pGA31R-MCS Rep_pl5A Cm tetR MCS DNA2.0

pGE21R-MCS Rep_c Km^K tetR P_T MCS T1

pJ248-mvaES Reppuc Ap Gent mvaES DNA2.0

pJ241- Rep_pUC Km^R Tl I M PM j PDs_c DNA2.0

MK.PMK.MPD.IDI

pUC57-ispS RepcoiEi Ap P[_ac

GenScript

pGB1004 Rep _E1 Km^R tetR i^spSpa,optEc Tl Glycos Biotechnologies, Inc. pGB1008 Rep_pl5A Cm^R tetR P_Lteto-i mvaE¾_fi0pfE(; Tl Glycos Biotechnologies, Inc. pGB1012 . Tl Glycos Biotechnologies, Inc. pGB1017 RepcoiEi Km tetR P_Lteto MK_Mj,optEc ispS; IDIEC Glycos Biotechnologies, Inc.

Tl

pGB1026 Glycos Biotechnologies, Inc. pGB1030 Rep_pl5A Cm tetR P_Lteto-i mvaESEf Tl P_Lteto Glycos Biotechnologies, Inc.

MKMj PMK_Ef MPD IDlEc Tl

pGB1033 Rep_pl5A Cm^R tetR P_Lteto-i mvaES_Ef pntAB_Ec Tl Glycos Biotechnologies, Inc.

MKMj PMKEf MPD Tl

Table 2 Notes: Ef = Enterococcus faecalis ATCC 700802; Mj = Methanocaldococcus jannaschii; Sc = Saccharomyces cerevisiae S288C; Ec = Escherichia coli MG1655; Pa = Populus alba; optEC = codon-optimized for expression in E. coli; MCS = multiple cloning site; Rep = origin of replication (ColEl, pl5A or pUC); PLteto-i = anhydrotetracycline- inducible promoter; Tl = terminator Tl of the rrnB operon of E. coli.

Table 3

GENE ABBREVIATIONS

Name Gene(s) Organism GenBank Reference

Acetoacetyl-CoA Thiolase/3- mvaE E. faecalis ATCC 700802 Accession: AF290092 Hydroxy-3-Methylghitaryl-CoA Protein ID: AAG02439.1 Reductase (bi-functional enzyme)

3-Hydroxy-3Methylglutaryl-CoA mvaS E. faecalis ATCC 700802 Accession: AF290092 Synthase ProteinJD: AAG02438.1

Mevalonate Kinase MK Methanocaldococcus jannaschii GenelD: 1451983

Protein ID: NP 248080.1

3-Hydroxy-3-Methylghitaryl-CoA

Reductase

HMG1 Arabidopsis ihaliana Accession: J04537 hmgA Haloferax volcanii Acession: NC_013967

ProteinJD: YP 003536599.

Hevea brasiliensis Accession: AY706757.1

HMGCR Homo sapiens GenelD: 3156

HMG1 Saccharomyces cerevisiae Accession: NM OO 1182434 mvaA Staphylococcus aureus Accession: AF290086

Hmgr Streptomyces sp. Strain CL190 Accession: ABO 13457 hmgA Sulfolobus solfataricus Accession: U95360

Phosphomevalonate Kinase PMK E. faecalis XYCC 700802 Gene Symbol: EF0902 Mevalonate Pyrophosphate MPD Saccharomyces cerevisiae

Decarboxylase

Isopentenyl Diphosphate Isomerase idi E. coli EcoCyc ID: G7508

Isoprene Synthase ispS Populus alba

Pyridine Nucleotide pntA E. coli EcoCyc ID: EG10744 Transhydrogenase, Membrane- pntB EcoCyc ID: EG10745 bound

Pyridine Nucleotide sthA E. coli EcoCyc ID: EG1 1428 Transhydrogenase, Soluble

[034] Plasmid pGA3 IR-MCS was constructed entirely by DNA synthesis by DNA2.0 (Menlo Park, CA), with the nucleotide sequence presented in Figure 3 (SEQ ID NO.: 1). Plasmid pGE21R-MCS was constructed by replacing the chloramphenicol resistance marker and the pl5A origin of replication of pGA3 IR-MCS with a kanamycin resistance marker and a ColEl origin. The sequence of pGE21R-MCS is presented in Figure 4 (SEQ ID NO.: 2).

[035] Plasmid pJ248-mv ES was constructed by DNA2.0 using the codon-optimized sequence of the mvaE and mvaS genes of Enterococcus faecalis ATCC 700802 (the codon- optimized sequences oimvaE and mvaS are as presented in Figure 5 (SEQ ID NO.: 3)). The mvaE and mvaS genes of Enterococcus faecalis ATCC 700802 were codon-optimized for expression in E. coli using the proprietary algorithms of DNA2.0, synthesized and inserted in the plasmid pJ248. Unique ribosomal binding sites were included in front of each gene, along with flanking endonuclease restriction sites for use in plasmid construction. [036] Plasmid pJ241-MK.PMK.MPD.IDI containing a codon-optimized synthetic operon was constructed entirely by DNA synthesis by DNA2.0, with the nucleotide sequence presented in Figure 6 (SEQ ID NO.: 4). The sequence of the synthetic operon, codon- optimized for expression in E. coli, encodes the mevalonate kinase gene of

Methanocaldococcus jannaschi, the phosphomevalonate kinase of Enterococcus faecalis ATCC 700802, the mevalonate pyrophosphate decarboxylase of Saccharomyces cerevisiae S288C, and the isopentenyl diphosphate isomerase gene of E. coli MG1655, including incorporated ribosomal binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

Plasmid pGB1004 was constructed by inserting a PCR product encoding the codon-optimized ispS gene of . alba into the Kpnl and Ncol restriction endonuclease sites of plasmid pGE21R-MCS (Figure 8). The PCR product encoding the ispS gene was amplified from the plasmid pUC57-ispS using AccuPrimer Pfx polymerase. Primer 1 incorporates a ribosomal binding site in front of the start codon of ispS and a Kpnl restriction site. Primer 2 incorporates an Ncol restriction site after the ispS stop codon. The PCR and plasmid pGE21R-MCS were digested with Kpnl and Ncol, followed by gel purification. The fragments were cloned together using standard cloning techniques. Figure 7 (SEQ ID NO.: 5) shows the codon-optimized sequence of the synthetic isoprene synthase gene oiPopulus alba, containing a ribosomal binding site and flanking restriction endonuclease sites used in subsequent cloning steps, but without the N-terminal transit peptide.

[037] Plasmid pGB 1012 was constructed by inserting a PCR product encoding the idi gene of E. coli into the Ncol site of pGB1004 (Figure 8). The PCR product encoding the idi gene was amplified from the plasmid pJ241-MK.PMK.MPD. IDI using AccuPrime Pfx polymerase. Primer 1 maintains the ribosomal binding site in front of the start codon of idi. Primer 1 and Primer 2 also include appropriate vector-overlapping 5' sequences for use with the In-Fusion Advantage PCR Cloning Kit (Clontech). The PCR product was gel-purified, as was pGB 1004 linearized with the restriction endonuclease Ncol. Fragments were directionally joined together using the In-Fusion cloning kit and GC5 competent cells, following the manufacturer's directions. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease- digested plasmid DNAs.

Plasmid pGB1017 was constructed by inserting a PCR product encoding the mevalonate kinase gene oiM. jannaschii into the Kpnl site of pGB1012 (Figure 8). The PCR product encoding the mevalonate kinase gene was amplified from plasmid pJ241-MK.PMK.MPD.IDI using AccuPrime Pfx polymerase. Primer 1 maintains the ribosomal binding site in front of the start codon of the mevalonate kinase gene. Primer 1 and Primer 2 also include appropriate vector-overlapping 5' sequences for use with the In-Fusion Advantage PCR Cloning Kit (Clontech). The PCR product was gel-purified, as was pGB1004 linearized with the restriction endonuclease Kpnl. Fragments were directionally joined together using the In- Fusion cloning kit and GC5 competent cells, following the manufacturer's directions.

Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

[038] Plasmid pGB 1008 was constructed by cloning the optimized mvaES genes from pJ248-mvaES into pGA31R-MCS as a KpnlVMluI DNA fragment using standard cloning techniques (Figure 9).

Plasmid pGB1026 was constructed by inserting an approximately 2,000 base pair PCR product encoding the pntAB genes of E. coli into the M site of pGB 1008 (Figure 9). The PCR product encoding the pntAB genes was amplified from genomic DNA of MG 1655 using AccuPrime Pfx polymerase. Primer 1 incorporates a ribosomal binding site in front of the start codon oipntA. Primer 1 and Primer 2 also include appropriate vector-overlapping 5' sequences for use with the In-Fusion Advantage PCR Cloning Kit (Clontech). The PCR product was gel-purified, as was pGB 1008 linearized with the restriction endonuclease M . Fragments were directionally joined together using the In-Fusion cloning kit and GC5 competent cells, following the manufacturer's directions. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

[039] Plasmid pGB1030 was created through the following process (Figure 10).

pGB1008 was digested with the restriction endonucleases Ncol and MM; the resulting 6.8 kb DNA fragment was gel-purified. Plasmid pJ241-MK.PMK.MPD.IDI was digested with the restriction endonucleases Ncol and MM; the resulting 4.1 kb containing the synthetic operon was gel-purified. The fragments were ligated together using the NEB Quick Ligation Kit (New England Biolabs) and transformed into GC5 competent cells. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs. [040] Plasmid pGB1033 was created through the following process (Figure 10).

pGB1026 was digested with the restriction endonucleases Ncol and SphI; the resulting 8.3 kb fragment was gel-purified. A second aliquot of pGB1026 was digested with the restriction endonucleases Mlul and SphI; the resulting 1.4 kb fragment was gel-purified. Plasmid pJ241-MK.PMK.MPD.IDI was digested with the restriction endonucleases Ncol and Mlul; the resulting 4.1 kb containing the synthetic operon was gel-purified. The fragments were ligated together in a trimolecular ligation reaction using the NEB Quick Ligation Kit (New England BioLabs) and transformed into GC5 competent cells. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

EXAMPLE 1

[041] Plasmids pGB1012 and pGB1030 were co-transformed into MG1655 using electroporation, generating the strain herein referred to as MG1655(pnt^~). This combination of plasmids provides a mevalonate-based pathway for production of isoprene. Plasmids pGB1012 and pGB 1033 were co-transformed into MG1655 using electroporation, generating the strain herein referred to as MG1655(pnt⁺). This combination of plasmids provides a mevalonate-based pathway for production of isoprene and overexpression of the PntAB transhydrogenase activity. Transformants were selected on LB Agar plates containing appropriate amounts of chloramphenicol and kanamycin.

[042] Seed cultures of MG1655(pnt^") and MG1655(pnt⁺) were prepared as follows: the cultures (stored as glycerol stocks at -80 °C) were used to inoculate 5 ml (LB medium as described above, containing appropriate amounts of chloramphenicol and kanamycin) seed cultures in 15 ml culture tubes and grown aerobically at 37 °C and 175 rpm for 16 hours. After 16 hours, the seed cultures were diluted into LB supplemented with appropriate antibiotics, 20 g/1 glucose, and 100 μg/l anhydrotetracycline to achieve an initial optical density of 0.3 at 600 nm. 10-ml aliquots of each diluted culture were placed in three 20-ml headspace vials; the diluted cultures were incubated at 37 °C and 175 rpm. At the end of 1, 2 and 3 hours of incubation, one of the headspace vials of each culture, MG1655(pnt^") or MG1655(pnt⁺), was removed from the shaking incubator, and the isoprene concentration was estimated by manually exposing a solid-phase microextraction fiber (85 μιη

Carboxen/PDMS) to sample the headspace. The fiber was desorbed at 300°C for 30 seconds prior to insertion into the headspace vial, exposed in the vial at ~37°C for 60 seconds to extract the volatiles, and immediately desorbed in the injector of an Agilent 5890 Series II GC at 200°C for 30 seconds (splitless injection, purge valve closed). The initial hold was at 30°C for 5 minutes, followed by a ramp at 20°C/min to 230°C, with a final hold of 2 minutes. The carrier gas was helium, the FID detector temperature was kept at 250°C, and the column was an Rtx-5 (30 m x 530 μιη x 3 μιη). The samples were compared to a commercial isoprene standard.

[043] The results of this example are presented in Figure 1 1. In the absence of overexpression of the transhydrogenase encoded by pntAB, E. coli MG1655(pnt^") engineered to produce isoprene via an NADPH-dependent HMG-CoA Reductase produced isoprene at approximately 0.02 mg/L per hour per OD600 of cell mass. Total isoprene accumulation reached 0.04 mg/L. In MG1655(pnt⁺), overexpression of the transhydrogenase encoded by pntAB improved both the rate of production and final isoprene titer approximately four- fold. The rate of production achieved greater than 0.08 mg/L per hour per OD600 of cell mass, while total isoprene accumulation reached 0.12 mg/L. EXAMPLE 2

[044] Plasmids pGB1017 and pGB1030 were co-transformed into FAOl using electroporation, generating the strain herein referred to as FA01(pnt^"). This combination of plasmids provides a mevalonate-based pathway for production of isoprene. Plasmids pGB1017 and pGB1033 were co-transformed into FAOl using electroporation, generating the strain herein referred to as FA01(pnt⁺). This combination of plasmids provides a mevalonate- based pathway for production of isoprene and overexpression of the PntAB transhydrogenase activity. Transformants were selected on LB Agar plates containing appropriate amounts of chloramphenicol and kanamycin.

[045] Seed cultures of FAOl(pnt ) and FA01(pnt⁺) were prepared as follows: the cultures (stored as glycerol stocks at -80 °C) were used to inoculate 5 ml (LB medium as described above, containing appropriate amounts of chloramphenicol and kanamycin) seed cultures in 15 ml culture tubes and grown aerobically at 37 °C and 175 rpm for 16 hours. After 16 hours, the seed cultures were diluted into LB supplemented with appropriate antibiotics, 40 mM MOPS, 20 g/1 glucose, and 200 μg/l anhydrotetracycline to achieve an initial optical density of 0.3 at 600 nm. 10-ml aliquots of each diluted culture were placed in three 20-ml headspace vials; the diluted cultures were incubated at 37 °C and 175 rpm. At the end of 1, 2 and 3 hours of incubation, one of the headspace vials of each culture, FAOl(pnt ) or FA01(pnt ), was removed from the shaking incubator, and the isoprene concentration was estimated by directly injecting headspace into an Agilent 7890A GC using an autosampler. First, the vial was incubated at 50°C with shaking for 1 minute. Then 1 mL of headspace was removed using a heated headspace syringe and injected into the GC inlet. The injector temperature was 250°C, the FID detector temperature was 300°C, the carrier was helium with a flow rate of 1 mL/min, and the split was 10: 1. The column was an HP-5 (30 m x 320 μιη x 0.25 μιη). The initial hold was at 30°C for 5 minutes, followed by a ramp at 2°C/min to 35°C, with no final hold. The samples were compared to a commercial isoprene standard.

[046] The results of this example are presented in Figure 12. In the absence of overexpression of the transhydrogenase encoded by pntAB, E. coli MFA01(pnt^") engineered to produce isoprene via an NADPH-dependent HMG-CoA Reductase produced isoprene at approximately 0.2 mg/L per hour per OD600 of cell mass. Total isoprene accumulation reached 0.6 mg/L. In FA01(pnt⁺), overexpression of the transhydrogenase encoded by pntAB improved both the rate of production and final isoprene titer. The rate of production achieved approximately 0.5 mg/L per hour per OD600 of cell mass, while total isoprene accumulation reached greater than 1.0 mg/L.

[047] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[048] While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

CLAIMS What is claimed is:

1. A non-naturally occurring microorganism that:

a. expresses a heterologous isoprene pathway in sufficient quantities to convert acetyl-CoA into isoprene, wherein the heterologous isoprene pathway comprises a 3-hydroxy-3-methylglutaryl-CoA reductase that uses NADPH as a co-factor; and

b. overexpresses a pyridine nucleotide transhydrogenase activity.

The non-naturally occurring microorganism of claim 1, wherein the 3-hydroxy-3- methylglutaryl-CoA reductase activity is encoded by the mvaE gene oi Enterococcus faecalis.

The non-naturally occurring microorganism of claim 1 or claim 2, wherein the pyridine nucleotide transhydrogenase is a membrane-bound pyridine nucleotide transhydrogenase.

The non-naturally occurring microorganism of claim 3, wherein the membrane-bound pyridine nucleotide transhydrogenase is encoded by the pntA and pntB genes of Escherichia coli.

5. The non-naturally occurring microorganism of claim 1 or claim 2, wherein the

pyridine nucleotide transhydrogenase is a soluble pyridine nucleotide

transhydrogenase.

6. The non-naturally occurring microorganism of claim 5, wherein the pyridine

nucleotide transhydrogenase is encoded by the sthA gene oi Escherichia coli.

7. The non-naturally occurring microorganism of claim 1, wherein the 3-hydroxy-3- methylglutaryl-CoA reductase uses NADPH, but not NADH, as a co-factor.

8. The non-naturally occurring microorganism of claim 1, wherein the 3-hydroxy-3- methylglutaryl-CoA reductase uses both NADPH and NADH as a co-factor.

9. The non-naturally occurring microorganism of claim 1, wherein the microorganism is derived from an Escherichia coli.

10. The non-naturally occurring microorganism of claim 9, wherein the heterologous genes are optimized for expression in Escherichia coli.

11. A method for producing isoprene, the method comprising:

a. culturing a non-naturally occurring microorganism that expresses a

heterologous isoprene pathway in sufficient quantities to convert acetyl-CoA into isoprene, wherein the heterologous isoprene pathway comprises a 3- hydroxy-3-methylglutaryl-CoA reductase that uses NADPH as a co-factor, and the strain further overexpresses a pyridine nucleotide transhydrogenase activity, in a culture medium thereby producing isoprene; and

b. recovering the isoprene.

12. The method of claim 9, wherein the overexpressed pyridine nucleotide

transhydrogenase of the non-naturally occurring microorganism is encoded by the pntA and pntB genes of Escherichia coli.

13. The method of claim 9, wherein the overexpressed pyridine nucleotide

transhydrogenase of the non-naturally occurring microorganism is encoded by the sthA gene of Escherichia coli.

14. The method of claim 10 or claim 1 1, wherein the 3-hydroxy-3-methylglutaryl-CoA reductase uses NADPH, but not NADH, as a co-factor.

15. The method of claim 10 or claim 1 1, wherein the 3-hydroxy-3-methylglutaryl-CoA reductase uses both NADPH and NADH as a co-factor.

16. The method of claim 11, wherein the microorganism is derived from an Escherichia coli.

17. The method of claim 16, wherein the heterologous genes are optimized for expression in Escherichia coli.