WO2008030995A2

WO2008030995A2 - Use of novel pepc to produce succinate

Info

Publication number: WO2008030995A2
Application number: PCT/US2007/077806
Authority: WO
Inventors: Ka-Yiu San; George N. Bennett; Mary Lou Harrison
Original assignee: Rice University
Priority date: 2006-09-06
Filing date: 2007-09-06
Publication date: 2008-03-13
Also published as: WO2008030995A8; WO2008030995A3

Abstract

A Leuconostoc mesenteroides phosphoenolpyruvate carboxylase enzyme has been identified that improves succinate production in engineered bacteria. Expression of the novel PEPC enzyme by engineered bacteria increases production and conversion of carbon to product is improved. Methods are described for making succinate using bacterial cultures expressing L. mesenteroides PEPC.

Description

USE OF NOVEL PEPC TO PRODUCE SUCCINATE

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to 60/824717 filed 09/06/2006. This application is hereby incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] The present invention was developed with funds from the United States Government. Therefore, the United States Government may have certain rights in the invention.

REFERENCE TO MICROFICHE APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] The invention relates to a novel phosphoenolpyruvate carboxylase enzyme that improves succinate production in engineered bacteria.

BACKGROUND OF THE INVENTION

[0005] The valuable specialty chemical succinate and its derivatives have extensive industrial applications. Succinic acid is used as a raw material for food, medicine, plastics, cosmetics, and textiles, as well as in plating and waste-gas scrubbing. Succinic acid can serve as a feedstock for such plastic precursors as 1 ,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. Further, succinic acid and BDO can be used as monomers for polyesters. If the cost of succinate can be reduced, it will become more useful as an intermediary feedstock for producing other bulk chemicals. Along with succinic acid, other 4-carbon dicarboxylic acids such as malic acid and fumaric acid also have feedstock potential.

[0006] The production of succinate, malate, and fumarate from glucose, xylose, sorbitol, and other "green" renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from non-renewable sources. Succinate is an intermediate for anaerobic fermentations by propionate -producing bacteria but those processes result in low yields and concentrations. Mixtures of acids are produced from E. coli fermentation. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product.

[0007] Manipulating enzyme levels through the amplification, addition, or reduction of a particular pathway can improve yields of a desired product. Various genetic improvements for succinic acid production under anaerobic conditions have been described that utilize the mixed-acid fermentation pathways of E. coli. In one example, the conversion of fumarate to succinate was improved by overexpressing native fumarate reductase (frd). Certain enzymes are not indigenous in E. coli, but can potentially help increase succinate production. By introducing pyruvate carboxylase (pyc), succinate production was enhanced. Other metabolic engineering strategies include inactivating competing pathways of succinate. When malic enzyme was overexpressed in a host with inactivated pyruvate formate lyase (pfl) and lactate dehydrogenase (IdK) genes, succinate became the major fermentation product. An inactive glucose phosphotransferase system iptsG) in the same mutant strain (Δpfl and Aldh) had also been shown to yield higher succinate production in E. coli and improve growth.

[0008] Overexpression of E. coli phosphoenolpyruvate carboxylases (PEPC) is another means that can be used to increase succinate production (Millard, et al. App. Environ. Microbiol. 62:1808-10 (1996)), and a variety of phosphoenolpyruvate carboxylases are known from various plant, bacterial, and eukaryotic sources. A 2004 journal article reported that a phosphoenolpyruvate carboxylase ipepc) from the archae bacteria, Methanothermobacter thertnautotrophicus had a novel structure and was about half the size of known bacterial and eukaryotic phosphoenolpyruvate carboxylases (1). The enzyme had unusual catalytic and regulatory properties; activity was not affected by acetyl coenzyme A, and it was about 50 times less sensitive to aspartate than the E. coli enzyme. [0009] Processes that use oxaloacetate as an intermediate, particularly those processes making use of glycolytic intermediates such as PEP to provide intermediates and/or product formation, are improved by the use of a more active PEPC. To further improve bacterial production, a novel PEPC protein has been identified and employed that increases the rate of product formation and product yield.

SUMMARY OF THE INVENTION

[0010] The present invention uses the phosphoenolpyruvate carboxylase gene ipepc) from Leuconostoc mesenteroides, which catalyzes the following reaction:

COO^{" "}

phosphoend- pynuvate oxatoacstat©

[0011] The enzyme is unexpectedly more active under anaerobic conditions and provides good conversion of initial carbon to final product. The L. mesenteroides pepc can be used in combination with engineered hosts to increase product levels thus improving conversion in engineered strains. The enzyme can be used to produce increased amounts of various compounds, including oxaloacetate, malate, fumarate, succinate, and compounds derived from them.

[0012] Bacterial cells engineered to produce elevated levels of succinate and other bioproducts are disclosed. Methods of using these cells to produce succinate are also disclosed. The engineered bacterial cells express recombinant L. mesenteroides PEPC increasing conversion of glucose to succinate. Production is greater than 1 mole succinate per mole glucose, more preferably greater than 1.1 moles succinate per mole glucose. These methods are useful for many engineered bacterial cells including E. coli, E. coli K-12, E. coli MG1655, E. coli SBS550MG, and £. coli SBS552MG. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1. Amino acid sequence of Leuconostoc mesenteroides PEPC (SEQ ID NO: 2).

[0014] FIG 2. Nucleotide sequence of Leuconostoc mesenteroides pepc (SEQ ID NO: I)-

[0015] FIG. 3. Multiple sequence alignment showing conserved amino acids with dots and identical amino acids with a star. Alignment performed with CLUSTAL W (1.83).

[0016] Fig. 4. Plasmids used for cloning and expression of PEPC proteins in E. coli.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0017] Carboxylic acids described herein can be a salt, acid, base, or derivative depending on structure, pH, and ions present. For example, the terms "succinate" and "succinic acid" are used interchangeably herein. Succinic acid is also called butanedioic acid (C₄H₆O₄). Chemicals used herein include formate, glyoxylate, lactate, malate, oxaloacetate (OAA), phosphoenolpyruvate (PEP), and pyruvate. Bacterial metabolic pathways including the Krebs cycle (also called citric acid, tricarboxylic acid, or TCA cycle) can be found in Principles of Biochemistry, by Lehninger as well as other biochemistry texts and in the NATIONAL LIBRARY OF MEDICINE^® PUBCHEM™ database (pubchem.ncbi.nlm.nih.gov) incorporated herein by reference.

[0018] The terms "operably associated" or "operably linked," as used herein, refer to functionally coupled nucleic acid sequences.

[0019] "Reduced activity" or "inactivation" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, or 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, and the like. By "null mutant" or "null mutation" what is meant is that activity is completely inactivated. In one example, the control plasmid is inserted without the gene of interest. In another example the gene of interest is completely removed by recombination. Additionally, the protein of interest may be removed by inactivation, mutation, or truncation which eliminates activity. Genes may be mutated individually or as an operon. For example, a single mutation may inactivate ackA and pta simultaneously, or the pathway may be mutated at either the ackA or pta gene to prevent formation of acetate.

[0020] "Overexpression" or "overexpressed" is defined herein to be greater than wild type activity, preferably above 125% increase, more preferably above 150% increase in protein activity as compared with an appropriate control species. Preferably, the activity is increased 100-500%. Overexpression is achieved by mutating the protein to produce a more active form, a more stable form, or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of a gene to the cell, up- regulating an existing gene, adding an exogenous gene, and the like.

[0021] The terms "disruption" and "disruption strains," as used herein, refer to cell strains in which the native gene or promoter is mutated, deleted, interrupted, or down- regulated in such a way as to decrease the activity of the gene. A gene is completely (100%) reduced by knockout or removal of part of or the entire genomic DNA sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can completely inactivate (100%) the gene product by completely preventing transcription and/or translation of active protein.

[0022] The term "exogenous" indicates that the protein or nucleic acid is a non-native molecule introduced from outside the organism or system, without regard to species of origin. For example, an exogenous peptide may be applied to the cell culture, an exogenous RNA may be expressed from a recombinant DNA transfected into a cell, or a native gene may be under the control of exogenous regulatory sequences.

[0023] As used herein "recombinant" is relating to, derived from, or containing genetically engineered material.

[0024] A gene or cDNA may be "optimized" for expression in E. coli, or other bacterial species using the codon bias for the species. Various nucleotides can encode a single peptide sequence. Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides which encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species.

[0025] In calculating "% identity" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-50. The default parameters were used, except the filters were turned OFF. As of Jan. 1, 2001 the default parameters were as follows: BLASTN or BLASTP as appropriate; Matrix = none for BLASTN, BLOSUM62 for BLASTP; G Cost to open gap default = 5 for nucleotides, 11 for proteins; E Cost to extend gap [Integer] default = 2 for nucleotides, 1 for proteins; q Penalty for nucleotide mismatch [Integer] default = -3; r reward for nucleotide match [Integer] default = 1; e expect value [Real] default = 10; W word size [Integer] default = 11 for nucleotides, 3 for proteins; y Dropoff (X) for blast extensions in bits (default if zero) default = 20 for blastn, 7 for other programs; X dropoff value for gapped alignment (in bits) 30 for blastn, 15 for other programs; Z final X dropoff value for gapped alignment (in bits) 50 for blastn, 25 for other programs. This program is available online at NCBI™ (www.ncbi.nlm.nih.gov/BLAST/).

[0026] Common restriction enzymes and restriction sites are found at NEB® (New England Biolabs®, www.neb.com) and Invitrogen® (www.invitrogen.com). ATCC®, American Type Culture Collection™ (www.atcc.org) has an extensive collection of cell strains that are publicly available and incorporated herein by reference. Abbreviations used throughout are listed in TABLE 1. TABLE 1 ABBREVIATIONS

Abbreviation Term

ACE acetate

LB Luria broth

Ap/ApR ampicillin / ampicillin resistance

ATCC® American Tissue -type Culture Collection

Cm/ Cm^R chloramphenicol /chloramphenicol resistance

ETOH ethanol

FORM formate

GLU glucose

Km/KmR kanamycin / kanamycin resistance

LAC lactate

NCBI™ National Center for Biotechnology Information

OAA oxaloacetate

PEP phosphoenolpyruvate

PEPC phosphoenolpyruvate carboxylase

PYC pyruvate carboxylase

PYR pyruvate

SUCC succinate

Wt wild-type adhE alcohol dehydrogenase ldhA lactate dehydrogenase iclR repressor of the glyoxylate operon ackA acetate kinase pta phosphotransacetylase sdhAB succinate dehydrogenase poxB pyruvate oxidase

[0027] Plasmids and strains used in certain embodiments of the invention are set f in TABLE 2.

TABLE 2 PLASMIDS AND STRAINS

Plasmid/Strain Genotype Ref pDHK29 Cloning vector Km^R Phillips, 2000 pGNB 10041 pTrcHisTOPO w/ L. mesenteroides pepc This work pGNB 10044 pDHK29 w/ L. mesenteroides pepc This work pHL413 pTrc99A w/ L. lactispyc Lin, 2004 pKK233-2 Expression vector w/ Trc promoter PHARMACIA^® pKK313 pKK233-2 w/ mutant Sorghum pepc Wang, 1992 pTrc99A Cloning vector Ap^R PHARMACIA^® pTrcHisTOPO Expression vector w/ Trc promoter, His Tag INVITROGEN^®

MG1655 Wild type (F-λ-) Guyer, 1988

Leuconostoc mesenteroides ATCC8293

SBS550MG AadhE-ldhA-iclR-ackA-pta (Cm^R) Sanchez, 2005

SBS552MG AadhE-ldhA-iclR-ackA-pta-sdhAB-poxB (Cm^R) Sanchez, 2005

ImPEPC SBS550MG (pGNB10041) This work

ImPEPC KmR SBS550MG (pGNB10044) This work sPEPC SBS550MG (pKK313) This work

PYC SBS550MG (pHL413) Lin, 2004

ImPEPC SBS552MG (pGNB10041) This work

ImPEPC-KmR SBS552MG (pGNB10044) This work sPEPC SBS552MG (pKK313) This work

[0028] When plasmids are used, the effect of host/plasmid interaction is identified by comparing three different systems consisting of: the host only, a plasmid expressing biologically active enzyme, and a control system with the expression vector alone. [0029] Cultures were started from glycerol stocks in 5 ml LB with antibiotics and grown aerobically overnight at 37°C with vigorous shaking.

[0030] In Experiment 1, each overnight culture was added to 400 ml LB with 35 μg/ml chloramphenicol (Cm³⁵), a combination of ampicillin, carbenicillin, and oxycillin at 67 μg/ml each, or 50 μg/ml kanamycin (Km⁵⁰). Cultures were grown aerobically 4-7 hours to an O.D.βoo of 1.5-4.

[0031] In Experiment 2, 400 ml cultures were grown aerobically for approximately 16 hours with either ampicillin at 200 μg/ml (Ap²⁰⁰) or Km⁵⁰ O.D.₆₀o readings were 2.3-2.8.

[0032] To begin the anaerobic culture phase, cells were pelleted and resuspended in a small volume of LB containing antibiotics at 1.1X concentrations of Cm^38'5, Ap²²⁰, or Km⁵⁵, and 1.1X OfNaHCO₃ at 2.2 g/L or 1.1 g/L.

[0033] Cell suspensions were adjusted to a concentration of 20 O. D. at 600nm and an aliquot of 9 mis was added to a 250 ml flask for each culture type. Each flask contained 0.5 g MgCO₃. Three flasks were set up for each culture type. Just before sparging with CO ., 1 ml of LB with 1OX glucose at 200 g/L and 1OX IPTG at 1OmM was added to each flask. Sparging was done for 1 to 1.5 minutes at 1 to 2L/min to create the anaerobic state. After sparging, flasks were sealed immediately with rubber caps and shaken at 37°C at 250 rpm. Reaction conditions were 20 O.D.6oo/ml, anaerobic, 110 mM glucose and 1 mM IPTG in LB.

[0034] Samples were taken at 23-25 hours and 46-53 hours for HPLC analysis.

[0035] The effect of genetic and environmental perturbations on metabolic and gene expression patterns was assessed by monitoring the extracellular metabolite concentrations and intermediate metabolite concentrations. Hexoses and fermentation products were measured by high-performance liquid chromatography (HPLC) using a specialized column (Vallino and Stephanopoulos 1993; Yang et al., 1999a). A BlO- RAD® AMINEX HPX-87H™ column specially designed for the analysis of small molecules was used to quantify chemical compounds. Fermentation products quantified include succinic acid, lactic acid, formic acid, acetic acid, pyruvate, glucose, and ethanol. In experiments where a detailed analysis is performed, the activities of certain key enzymes in the metabolic pathways can be measured. EXAMPLE 1: CLONING OF NOVEL PEPC GENE

[0036] A BLAST comparison of M. thermautotrophicus D69226 with the genomic sequence of the lactic acid bacteria Leuconostoc mes enter oides at GenBank Ace. No. NC 008531 (aa 1667466-1668980) revealed a region homologous to the M. thermautotrophicus pepc. A culture of L. mesenteroides was obtained from the American Type Culture Collection (ATCC: ATCC: 8293). The organism was grown in LB with 4% glucose at 26°C, shaking at 200 rpm. Genomic DNA was extracted from a cell pellet using a PUREGENE™ DNA Purification System (GENTRA^®, part no. D-6000A) following the gram-positive protocol.

[0037] PCR of the pepc gene found at COGl 892 at GenBank Ace. No. NZ AABH 02000008, region 43258-44772 (SEQ ID NO: 1 & 2) was performed using primers LmpepcF (SEQ ID NO: 3; ATG ACA TCA CGT AAA ATC CCT TC) and LmpepcR (SEQ ID NO: 4; TTATCC AAG GAA GTG TCG TAA TTG) which amplified the gene from the start codon through the stop codon. PCR was performed with MASTERTAQ™ enzyme (EPPENDORF^® kit 0032 002.552) and the 1515bp fragment was confirmed by gel electrophoresis. The PCR product was inserted into pTrcHisTOPO (INVITROGEN^®) which is 4.4 kb, resulting in pGNB 10041, a 5.9 kb vector expressing the L. mesenteroides PEPC with the trc promoter inducible with IPTG.

[0038] This PCR-amplified pepc gene was sequenced, translated and compared by homology search against other PEPC sequences. The closest sequence was the Leuconostoc mesenteroides (ATCC: 8293) which had a difference of three base pairs from the PEPC described herein (see NC 008531 REGION: 1667466-1668980 and YP 819158.1, Oct. 2006). Two of the base pair substitutions did not result in a change in the amino acid sequence; the third substitution resulted in a change from alanine to threonine at position 40. Thus there can be either a c or a t at position 51, an a or a g at position 118, or a t or a c at position 1164. The next closest homolog was EAV38804.1 of Oenococcus oeni ATCC: BAA-1163 with 307/505 (60%) identity and YP 811302.1 of Oenococcus oeni PSU-I, also having 307/505 (60%) identity. A multiple alignment of the sequences showing conserved residues is found in Fig. 3A and 3B. The alignments indicate which residues cannot be changed; however a small number of conservative changes in the remaining residues resulting in 99, 98, 97, 96 or 95% amino acid identity are expected to result in a functional PEPC. [0039] A second vector containing the L. mes enter oides pepc gene was constructed by digesting pGNB 10041 with BamHI and EcoRI, and inserting the gel-purified 1.5 kb fragment into BamHI/EcoRI-digested pDHK29 (4.2 kb) (Phillips, 2000) to yield pGNB 10044 (5.7 kb) which contains an IPTG inducible lac promoter.

[0040] The vector pHL413, which is a construct from Lin (2004) containing the Lactococcus lactis pyruvate carboxylase (pyc) gene under the control of a pTrc promoter, was used for comparison purposes since it has been shown to result in increased succinate production. Control vectors pTrc99A (PHARMACIA^®) and pDHK29 were also included; pTrc99A is the base vector for pHL413 and pDHK29 is the control base vector for pGNB 10044. The pKK313 plasmid contains the mutant Sorghum pepc gene under the control of a trc promoter (Wang, 1992). Plasmid maps are found in Figure 4. Relevant features of the plasmids are summarized in Table 1.

EXAMPLE 2: SUCCINATE PRODUCTION WITH PEPC

[0041] The HPLC data show that, in the SBS550MG host, the pGNB 10041 vector containing the Leuconostoc pepc gene gave the highest yield of succinate. Acetate and formate were significant byproducts in spite of the relevant host gene inactivations; a yield of 1.3 moles succinate/mole glucose (1.3 xl lO mM or 144 mM) was achieved. There were no dramatic changes in the concentrations of the measured metabolites from the 23 hours to the 46-48 hour samples.

[0042] The host alone showed 120 mM succinate as product, for a yield of 1.1 moles succinate/mole glucose in 23 hours.

TABLE 3 HPLC RESULTS (IN MILLIMOLAR)

23 hours (2 g/L NaHCO₃) GLU SUCC LAC FORM ACE ETOH PYR

ImPEPC AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pGNB 10041)) 0 144 2 14 29 5 0

ImPEPC(Cm¹*) AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pGNB 10044)) 38 71 2 7 10 1 0

Vector control AadhE-ldhA-iclR-ackA-pta

(SBS550MG(ptrc99A)) 0 104 8 23 38 6 0

Vector control (Cm^R) AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pDHK29)) 17 105 J 15 20 2 0

Host Control AadhE-ldhA-iclR-ackA-pta (SBS550MG) 0 123 2 24 28 24 0

46-48 hours(2 g/L NaHCO₃) GLU SUCC LAC FORM ACE ETOH PYR TABLE 3 HPLC RESULTS (IN MILLIMOLAR)

ImPEPC AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pGNB 10041)) 0 144 4 10 31 5 0

ImPEPC(Cm¹*) AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pGNB 10044)) 23 88 1 8 15 2 0

Vector control AadhE-ldhA-iclR-ackA-pta

(SBS550MG(ptrc99A)) 0 104 8 20 40 7 0

Vector control (Cm^R) AadhE-ldhA-iclR-ackA-pta

(SBS550MG(pDHK29)) 2 123 8 8 26 J 0

Host Control AadhE-ldhA-iclR-ackA-pta

(SBS550MG) 0 120 5 22 28 25 0

[0043] The ImPEPC was approximately 20% more effective at producing succinate, increasing both the rate of succinate production and the amount of succinate produced. The host and vector controls produce succinate at a much slower rate and do not convert all of the glucose to into product. The host controls actually produce more ethanol and formate than the ImPEPC cells. Increasing both rate and yield are essential for improving large scale bioreactor production of succinate.

EXAMPLE 3: OPTIMIZING SUCCINATE PRODUCTION

[0044] The SBS550MG(pHL413), PYC, strain routinely results in the maximum theoretical production of 176 mM succinate (for a yield of 1.6) in 24 hours by use of the pyruvate carboxylase. A fresh stock of SBS550MG(pHL413) was obtained and used in Experiment 2; at 25 hours this culture showed 155 mM succinate or a yield of 1.4 whereas at 53 hours the yield was up to 1.6. This culture served as a positive control for comparison purposes.

[0045] The SBS552MG, host control, and SBS552MG (pKK313), vector control, cultures showed a very low production of succinate and incomplete use of glucose even at 53 hours. The SBS552MG (pGNB10041), ImPEPC, culture used all of the glucose by 25 hours, with an eventual yield of 1.1 moles succinate/mole glucose at 53 hours.

TABLE 4 SUCCINATE PRODUCTION WITH (1 G/L NAHCO₃)

4 hours GLU SUCC LAC FORM ACE ETOH PYR

Host Control AadhE-ldhA-iclR-ackA-pta-sdhAB-

74 10 3 13 poxB (SBS552MG) sPΕP C J adhE-ldhA-iclR-ackA-pta-sdhAB-poxB

(SBS552MG (pKK313)) 71 13 5 0

ImPEPC Δ adhE-ldhA-iclR-ackA-pta-sdhAB-poxB

(SBS552MG (pGNB10041)) 52 44 16 0

PYC AadhE-ldhA-iclR-ackA-pta

(SBS550MG (pHL413)) 48 65 3 0

25 hours GLU SUCC LAC FORM ACE ETOH PYR

Host Control ΔadhE-ldhA-iclR-ackA-pta-sdhAB-

66 12 10 23 poxB (SBS552MG) sPΕP C J adhE-ldhA-iclR-ackA-pta-sdhAB-poxB 65 17 10 1 TABLE 4 SUCCINATE PRODUCTION WITH (1 G/L NAHCO₃)

(SBS552MG (pKK313))

ImPEPC A adhE-ldhA-iclR-ackA-pta-sdhAB-poxB r. , ΛΠ , -. , , q , η

(SBS552MG (pGNB10041))

PYC S.adhE-ldhA-iclR-ackA-pta ₀ , ^ _{0 9 9 0 0}

(SBS550MG (pHL413)) _ _

53 hours GLU SUCC LAC FORM ACE ETOH PYR

Host Control A adhE-ldhA-iclR-ackA-pta-sdhAB- poxB (SBS552MG) sPEPC AadhE-ldhA-iclR-ackA-pta-sdhAB-poxB _{7ή 1 Q} , _{1 Λ} , , , _<■

(SBS552MG (pKK313)) 76 19 1 10 13 3 5 imPΕP C A adhE-ldhA-iclR-ackA-pta-sdhAB-poxB _{0 1 24 1} ,_{7 22 8 5}

(SBS552MG (pGNB10041))

PYC AadhE-ldhA-iclR-ackA-pta n 1 74 fi 1 ^? fi fi

(SBS550MG (pHL413)) '

[0046] L. mesenteroides PEPC improved both the rate of succinate production and the amount of succinate produced per mole glucose as compared with the PEPC from sorghum. Thus using L. mesenteroides PEPC dramatically improved product formation. Additionally, ImPEPC was robust improving production in a number of cell backgrounds and under a variety of conditions. Thus ImPEPC provides another useful tool for increasing succinate production in bacteria.

REFERENCES:

[0047] All references are listed herein for the convenience of the reader. Each is incorporated by reference in its entirety.

1. Patel, et al., "The phosphoenolpyruvate carboxylase from Methanothermobacter thermautotrophicus has a novel structure." J. Bacteriol. 186:5129-37 (2004).

2. Phillips, et al., "High copy number plasmids compatible with commonly used cloning vectors r BioTechniques 28: 400-8 (2000).

3. Lin, et al., "Increasing the acetyl-CoA pool in the presence of overexpressed phosphoenolpyruvate carboxylase or pyruvate carboxylase enhances succinate production in Escherichia coli " Biotechnol. Prog. 20:1599-604 (2004).

4. Wang, et al., "Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4 phosphoenolpyruvate carboxylase from Sorghum." J. Biol. Chem. 267: (1992)

5. Sanchez, et al., "Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity" Metab. Eng. 7:229-39 (2005).

Claims

What is claimed is:

1. A bacteria comprising an expression vector encoding Leuconostoc mes enter oides phosphoenolpyruvate carboxylase (PEPC) protein, and having reduced activity of alcohol dehyrodgenase, lactate dehydrogenase, acetate operon repressor, acetate kinase and phosphotransacetylase.

2. The bacteria of claim 1, wherein expression of said PEPC increases conversion of glucose to succinate.

3. The bacteria of claim 1, wherein expression of said PEPC increases succinate production to greater than 1 mole succinate per mole glucose.

4. The bacteria cell of claim 1, wherein expression of said PEPC increases succinate production to greater than 1.1 moles succinate per mole glucose.

5. The bacteria cell of claim 1, wherein said PEPC comprises SEQ ID NO: 2.

6. The bacteria cell of claim 1, wherein said PEPC consists of SEQ ID NO: 2.

7. A bacteria comprising a expression vector encoding Leuconostoc mesenteroides PEPC protein having 95% identity to the amino acid sequence of SEQ ID NO: 2 and wherein said PEPC functions to convert phosphoenolpyruvate to oxaloacetate.

8. The bacteria of claim 8, wherein the PEPC protein has 99% identity to the amino acid sequence of SEQ ID NO: 2.

9. The bacteria of claim 8, wherein the PEPC protein comprises SEQ ID NO: 2.

10. The bacteria of claim 7, wherein said PEPC protein consists of SEQ ID NO: 2.

11. A method of producing succinate comprising:

a) culturing a bacteria comprising an expression vector encoding Leuconostoc mesenteroides PEPC and having reduced activity of alcohol dehyrodgenase, lactate dehydrogenase, acetate operon repressor, acetate kinase and phosphotransacetylase under conditions suitable for the production of succinate; and

b) isolating succinate from said bacteria.

12. The method of claim 7, wherein expression of said L. mesenteroides PEPC increases succinate production to greater than 1 mole succinate per mole glucose.

13. The method of claim 7, wherein expression of said L. mesenteroides PEPC increases succinate production to greater than 1.1 moles succinate per mole glucose.

14. The method of claim 7, wherein said bacterial cell is selected from the group consisting of E. coli, E. coli K-12, E. coli MG1655, E. coli SBS550MG, and E. coli SBS552MG.

15. The method of claim 7, wherein said recombinant DNA encodes Leuconostoc mesenteroides PEPC of SEQ ID NO: 2.

16. The method of claim 7, wherein said recombinant DNA encodes Leuconostoc mesenteroides PEPC that has 95% homology to SEQ ID NO: 2.

17. The method of claim 7, wherein said recombinant DNA is SEQ ID NO: 1.