US20120058530A1 - Engineering the pathway for succinate production - Google Patents

Engineering the pathway for succinate production Download PDF

Info

Publication number
US20120058530A1
US20120058530A1 US13/256,460 US201013256460A US2012058530A1 US 20120058530 A1 US20120058530 A1 US 20120058530A1 US 201013256460 A US201013256460 A US 201013256460A US 2012058530 A1 US2012058530 A1 US 2012058530A1
Authority
US
United States
Prior art keywords
succinate
production
pck
glucose
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/256,460
Other languages
English (en)
Inventor
Xueli Zhang
Kaemwich Jantama
Jonathan C. Moore
Laura R. Jarboe
Keelnatham T. Shanmugam
Lonnie O'Neal Ingram
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Florida Research Foundation Inc
Original Assignee
University of Florida Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Florida Research Foundation Inc filed Critical University of Florida Research Foundation Inc
Priority to US13/256,460 priority Critical patent/US20120058530A1/en
Assigned to UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC. reassignment UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZHANG, XUELI, MOORE, JONATHAN C., JANTAMA, KAEMWICH, JARBOE, LAURA R., INGRAM, LONNIE O., SHANMUGAM, KEELNATHAM T.
Publication of US20120058530A1 publication Critical patent/US20120058530A1/en
Assigned to ENERGY, UNITED STATES DEPARTMENT OF reassignment ENERGY, UNITED STATES DEPARTMENT OF CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF FLORIDA
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C12N9/1211—Thymidine kinase (2.7.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01001—Pyruvate carboxylase (6.4.1.1)

Definitions

  • the present invention is in the field of production of succinic acid from renewable biological feedstocks using microbial biocatalysts.
  • This invention discloses the genetic modifications to the biocatalysts that are useful in achieving high efficiency for succinic acid production. More specifically, this invention provides genetically modified biocatalysts that are suitable for the production of succinic acid from renewable feedstocks in commercially significant quantities.
  • a 2004 U.S. Department of Energy report entitled “Top value added chemicals from biomass” has identified twelve building block chemicals that can be produced from renewable feed stocks.
  • the twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric and maleic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol.
  • the fermentative production of these building block chemicals from renewable feedstocks will become increasingly competitive as petroleum prices in crease.
  • These building block chemicals are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. These twelve building block chemicals can be subsequently converted to a number of high-value bio-based chemicals or materials.
  • succinate can serve as a substrate for transformation into plastics, solvents, and other chemicals currently made from petroleum (Lee et al., 2004; Lee et al., 2005; McKinlay et al., 2007; Wendisch et al., 2006; Zeikus et al., 1999).
  • Many bacteria have been described with the natural ability to produce succinate as a major fermentation product (Table 1). However, complex processes, expensive growth media and long incubation times are often required to produce succinic acid from these naturally occurring succinic acid producing microorganisms.
  • E. coli strain NZN111 produced 108 mM succinate with a molar yield of 0.98 mol succinate per mol of metabolized glucose (Chatterjee et al., 2001; Millard et al., 1996; Stols and Donnelly, 1997). This strain was engineered by inactivating two genes (pflB encoding pyruvate-formate lyase and ldhA encoding lactate dehydrogenase), and over-expressing two E.
  • E. coli strain HL27659k was engineered by mutating succinate dehydrogenase (sdhAB), phosphate acetyltransferase (pta), acetate kinase (ackA), pyruvate oxidase (poxB), glucose transporter (ptsG), and the isocitrate lyase repressor (iclR).
  • succinate dehydrogenase sdhAB
  • pta phosphate acetyltransferase
  • ackA acetate kinase
  • poxB pyruvate oxidase
  • ptsG glucose transporter
  • iclR isocitrate lyase repressor
  • This strain produced less than 100 mM succinate and required oxygen-limited fermentation conditions (Cox et al., 2006; Lin et al., 2005a, 2005b, 2005c; Yun et al., 2005). Analysis of metabolism in silica has been used to design gene knockouts to create a pathway in E. coli that is analogous to the native succinate pathway in Mannheimia succiniciproducens (Lee et al., 2005 and 2006). The resulting strain, however, produced very little succinate. Andersson et al. (2007) have reported the highest levels of succinate production by engineered E. coli (339 mM) containing only native genes.
  • Rhizobium eteloti pyruvate carboxylase (pyc) was over-expressed from a multicopy plasmid to direct carbon flow to succinate.
  • pyc Rhizobium eteloti pyruvate carboxylase
  • Strain SBS550MG was constructed by inactivating the isocitrate lyase repressor (iclR), adhE, ldhA, and ackA, and over-expressing the Bacillus subtilis citZ (citrate synthase) and R. etli pyc from a multi-copy plasmid (Sanchez et al.; 2005a). With this strain, 160 mM succinate was produced from glucose with a molar yield of 1.6.
  • succiniciproducens were combined for the production of succinic acid from glucose at high yield, titer and productivity (Meynial-Salles et al., 2007).
  • E. coli The majority by far of scientific knowledge of E. coli is derived from investigations in complex medium such as Luria broth rather than mineral salts medium using low concentrations of sugar substrates (typically 0.2% w/v; 11 mM) rather than the 5% (w/v) glucose (278 mM) and 10% (w/v) glucose (555 mM) used in the studies reported herein. Large amounts of sugar are required to produce commercially significant levels of product.
  • Previous researchers have described the construction of many E. coli derivatives for succinate production in complex medium (Table 1). With complex medium, rational design based on primary pathways has been reasonably successful for academic demonstrations of metabolic engineering. However, the use of complex nutrients for production of bacterial fermentation products increases the cost of materials, the cost of purification, and the cost associated with waste disposal. Use of mineral salts medium without complex media components should be much more cost-effective.
  • E. coli C grows well in NBS mineral salts medium containing glucose and produces a mixture of lactate, acetate, ethanol and succinate as fermentation products ( FIG. 1A ; Table 4).
  • Table 1 the studies reported herein have focused on the development of strains that are able to convert high level of sugars into succinate using mineral salts medium to minimize the costs of materials, succinate purification, and waste disposal.
  • One aspect of the invention provides various strains of E. coli , that produce succinate at high titers and yields in mineral salts media during simple, pH-controlled, batch fermentations without the need for heterologous genes or plasmids.
  • the inventors have surprisingly identified a number of target genes useful in genetic manipulation of biocatalysts for achieving high efficiency for succinic acid production.
  • the method for obtaining the biocatalysts for succinic acid production from biological feedstocks combines rational genetic manipulations and the process of metabolic evolution.
  • the mutation of the genes in the bacterial chromosome is accomplished without introducing any exogenous genetic material.
  • the mutation of the endogenous genes are accomplished either by introducing point mutation or by introducing a stop codon in the open reading frame of the endogenous gene.
  • the entire open reading frame of the endogenous gene is deleted from the chromosomal DNA.
  • the expression of certain endogenous genes is significantly increased.
  • the transcription of the endogenous gene is increased by means of introducing certain mutations in the promoter region of the endogenous genes.
  • the transcription of the endogenous gene is enhanced by means of reliving the repressive control of the target gene.
  • an exogenous nucleotide sequence may be introduced to inactivate a target gene for the purpose of selecting a bacterial strain with a mutated gene with desirable phenotype.
  • the exogenous nucleotide sequence introduced into the microbial genome is subsequently removed in a seamless fashion without leaving behind any residual exogenous nucleotide sequence.
  • the rationally designed genetic manipulations can all be done in a single stage or in multiple stages in which a single genetic change is accomplished at one time.
  • the microbial strain resulting from genetic manipulations may be subjected to metabolic evolution in order to improve the yield, titer and volumetric productivity of the desired organic acid.
  • the rational genetic manipulations are done in stages and the process of metabolic evolution is carried out in between the stages of genetic manipulation.
  • the spontaneous mutations that occur during the metabolic evolution are identified through sequencing appropriate regions of the chromosomal DNA. In yet another embodiment of the present invention, the mutations that occur during the metabolic evolution are identified by measuring the activities of suspect enzyme.
  • one or more of the genes coding for the proteins known to function in the fermentative pathways are inactivated through one or more mutations.
  • the genes which are functional homologues of the genes coding for the proteins functioning in the fermentative pathway are inactivated beside the genes coding for the proteins directly involved in the fermentative pathway.
  • the genes functioning within the TCA cycle are genetically manipulated so that there is an increased flow of carbon towards succinic acid production.
  • the carbon flow through reductive arm of the TCA cycle is enhanced.
  • the carbon flow through oxidative arm of the TCA cycle is genetically manipulated.
  • the carbon flow to succinic acid is improved through genetic manipulation of glyoxalate bypass pathway closely associated with TCA cycle.
  • the carbon flow from TCA to other metabolic pathways within the cell is blocked so that the carbon pool within the cell is funneled towards the succinic acid production.
  • the carbon flow into the TCA cycle is enhanced through genetic manipulation leading to one of more carboxylating enzymes within the cell.
  • the expression of one or more carboxylating enzyme is achieved through the genetic manipulation of the promoter region or by relieving the repression of the gene expression.
  • the phosphoenol pyruvate pool within the cell is conserved by mutating those genes involved in the carbon uptake pathway requiring phosphoenol pyruvate.
  • the phosphotransferase system for carbon uptake is inactivated in order to conserve the phosphoenol pyruvate available for the operation of the TCA cycle.
  • the present invention illustrates a number of targets that can be genetically manipulated to achieve an increased succinic acid production. All these various targets described in the present invention can be genetically manipulated to achieve an improved succinic acid production. In the most preferred embodiment, a minimum number of targets are selected for genetic manipulation to achieve a desirable rate of succinic acid production.
  • the biocatalysts are selected for their ability to produce succinic acid at high titer, yield and volumetric productivity.
  • a biocatalyst capable of producing at least 1.0 mole of succinic acid for every one mole of carbon source consumed is preferred.
  • the biocatalyst is selected during metabolic evolution for its ability to produce at least 1.0 mole of succinic acid for every mole of carbon source consumed in a mineral salt medium and coupling the succinic acid production to microbial growth.
  • biocatalysts capable of producing succinic acid using glycerol as a feed stock are provided.
  • FIGS. 1A-1B Fermentation of glucose to succinate.
  • FIG. 1A shows the standard pathway for fermentation of glucose by E. coli . This pathway has been redrawn from Unden and Kleefeld (2004).
  • Bold arrows represent central fermentative pathways.
  • Crosses represent the gene deletions performed in this study to engineer KJ012 (ldhA, adhE, ackA).
  • Genes and enzymes ldhA, lactate dehydrogenase; pflB, pyruvate-formate lyase; focA, formate transporter; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB, and fumC, fumarase isozymes; frdBCD, fumarate reductase; fdh, formate dehydrogenase; icd, isocitrate dehydrogenase; acs, acetyl ⁇ CoA synthetase; mgsA, methylglyoxal synthase; poxB,
  • FIG. 1B shows the coupling of ATP production and growth to succinate production in engineered strains of E. coli for a standard pathway for glucose fermentation.
  • Solid arrows connect NADH pools.
  • Dotted arrows connect NAD + pools.
  • growth is obligatory coupled to the production of ATP and the oxidation of NADH.
  • FIGS. 2A-2D Potential carboxylation pathways for succinate production by E. coli . Genes encoding key carboxylating enzymes are shown in bold.
  • FIG. 2A shows the reaction catalyzed by phosphoenolpyruvate (PEP) carboxylase enzyme (PPC). No ATP is produced from the carboxylation of phosphoenolpyruvate (PEP) by the PPC enzyme. This is regarded as the primary route for succinate production by E. coli during glucose fermentation.
  • FIG. 2B shows the NADH-dependent malic enzyme. Energy is conserved during the production of ATP from ADP and PEP by pyruvate kinase (pykA or pykF).
  • FIG. 2C shows the NADPH-dependent malic enzyme. Energy is conserved during the production of ATP from ADP and PEP by pyruvate kinase (pykA or pykF).
  • Malic enzyme (maeB) catalyzes an NADPH-linked, reductive carboxylation to produce malate.
  • FIG. 2D shows the reaction catalyzed by PEP carboxykinase (PCK). Energy is conserved by the production of ATP during the carboxylation of PEP to produce oxaloacetic acid.
  • PCK PEP carboxykinase
  • FIGS. 3A-3C Growth during metabolic evolution of KJ012 to produce KJ017, KJ032, and KJ060.
  • Strain KJ012 was sequentially transferred in NBS medium containing 5% (w/v) ( FIG. 3A ) and 10% (w/v) ( FIG. 3B ) glucose, respectively to produce KJ017.
  • the resulting strain was initially subcultured in medium supplemented with acetate ( FIG. 3C ). Acetate levels were decreased and subsequently eliminated during further transfers to produce KJ060.
  • FIGS. 4A-4F Summary of fermentation products during the metabolic evolution of strains for succinate production. Cultures were supplemented with sodium acetate as indicated. Black arrows represent the transition between fermentation conditions as indicated by text. No formate and only small amounts of lactate were detected during metabolic evolution of KJ032. No formate and lactate were detected during metabolic evolution of KJ070 and KJ072. Metabolic evolution of KJ012 to KJ017 in the medium containing 5% w/v glucose ( FIG. 4A ) and in the medium containing 10% w/v glucose ( FIG. 4B ). Metabolic evolution of KJ032 to KJ060 in a medium containing 5% w/v glucose ( FIG. 4C ) and 10% w/v glucose ( FIG.
  • FIG. 4D Metabolic evolution of KJ070 to KJ071 in the medium containing 10% glucose
  • FIG. 4E Metabolic evolution of KJ072 to KJ073 in the medium containing 10% glucose
  • FIG. 4F Symbols for FIG. 4A-4F : ⁇ , succinate; ⁇ , formate; ⁇ , acetate; ⁇ , malate; ⁇ , lactate; and ⁇ , pyruvate.
  • FIG. 5 Diagram summarizing steps in the genetic engineering and metabolic evolution of E. coli C as a biocatalyst for succinate production. This process represents 261 serial transfers providing over 2000 generations of growth-based selection. Clones were isolated from the final culture of each regimen and assigned strain designations, shown in parenthesis in Table 4.
  • FIG. 6 Standard pathway for the fermentation of glucose-6-phosphate with associated pathways showing the genes that have been deliberately deleted in constructs engineered for succinate production.
  • Solid arrows represent central fermentative pathways.
  • Dashed arrow represents the microaerophilic pathway for the oxidation of pyruvate to acetate.
  • Dotted arrows show pathways including glyoxylate bypass that normally function during aerobic metabolism. Boxed crosses represent the three initial gene deletions (ldhA, adhE, ackA) that were used to construct KJ012 and KJ017.
  • KJ032 (ldhA, adhE, ackA, focA, pflB), and KJ070 (ldhA, adhE, ackA, focA, pflB, mgsA), and KJ072 (ldhA, adhE, ackA, focA, pflB, mgsA, poxB).
  • Genes and enzymes ldhA, lactate dehydrogenase; focA, formate transporter; pflB, pyruvate-formate lyase; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB, and fumC, fumarase isozymes; frdABCD, fumarate reductase; fdh, formate dehydrogenase; mgsA, methylglyoxal synthase; gloAB, glyoxylase I and II; poxB, pyruvate oxidase; aceA, isocitrate lyas
  • FIGS. 7A-7C Production of succinate and malate in mineral salts media with 10% glucose (w/v) by derivatives of E. coli C.
  • FIG. 7A shows succinate production by KJ060 in AM1 medium.
  • FIG. 7B shows succinate production by KJ073 in AM1 medium.
  • FIG. 7C shows production of malate by KJ071 in NBS medium. Fermentations were inoculated at a level of 33 mg DCW l ⁇ 1 .
  • Symbols for FIG. 7A-7C ⁇ , glucose; ⁇ , succinate; ⁇ , malate; ⁇ , cell mass.
  • FIG. 8 Steps involved in the construction of plasmid pLOI4162.
  • Short solid arrows associated with pEL04 and pLOI4152 represent primers used for DNA amplification.
  • FIG. 9 Succinate production from glucose-6-phosphate in KJ073.
  • the pck gene encoding phosphoenolpyruvate carboxykinase, the primary carboxylating enzyme involved in succinate production in this study, is shown in reverse type.
  • Solid arrows indicate reactions expected to be functional during anaerobic fermentation of glucose.
  • Solid crosses indicate deleted genes. Boxed crosses represent key deletions used to construct initial strain for succinate production, KJ017 (ldhA, adhE, ackA).
  • the dashed line represents oxidation of pyruvate to acetate by PoxB, a process that is typically functional only under microaerophilic conditions.
  • the dotted lines indicate reactions that are primarily associated with aerobic metabolism.
  • Genes and enzymes ldhA, lactate dehydrogenase; pflB, pyruvate-formate lyase; focA, formate transporter; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; pck, phosphoenolpyruvate carboxykinase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB, and fumC, fumarase isozymes; frdABCD, fumarate reductase; fdh, formate dehydrogenase; icd, isocitrate dehydrogenase; acs, acetyl ⁇ CoA synthetase; mgsA, methylglyoxal synthase; po
  • tdcE gene pyruvate formate-lyase, homologous to pflB
  • tcdD gene propionate kinase, homologous to ackA
  • FIG. 10 Expanded portion of metabolism illustrating the pathways of additional genes that have been deleted (solid crosses).
  • Succinate and acetate are principal products (boxed) from KJ073 fermentations.
  • Genes and enzymes citDEF, citrate lyase; gltA, citrate synthase; aspC, aspartate aminotransferase; pck, phosphoenolpyruvate carboxykinase; sfcA, NAD + -linked malic enzyme; fumA & fumB, fumarase; frdABCD, fumarate reductase; pykA & pykF, pyruvate kinase; tdcE, pyruvate formate-lyase (homologue of pflB); pta, phosphate transacetylase; tcdD, acetate kinase (homologue of ackA).
  • FIG. 11 Glucose fermentation in E. coli strains engineered for succinate production.
  • the 5 solid stars indicate metabolic steps that have been blocked by constructing deletions.
  • the 2 open stars indicate metabolic steps that have been blocked by mutations acquired during metabolic evolution.
  • Dotted arrows indicate non-functional or weakly functional activities in succinate-producing mutants (KJ060 and KJ073).
  • Bold arrows in the vertical direction indicate the primary pathway for succinate production in KJ060 and KJ073. This pathway is functionally equivalent to that of succinate-producing rumen bacteria.
  • the two genes galP and pck were transcriptionally activated during strain development.
  • GalP was subsequently found to serve as the primary transporter for glucose with ATP-dependent phosphorylation by glucokinase (glk).
  • FIGS. 12A-12C Comparison of transcript abundance of the engineered succinate-producing strains.
  • FIG. 12A shows the relative abundance of transcripts for the genes pck, ppc, sfcA and maeB in ATCC8739, KJ012, KJ017, KJ060, KJ071 and KJ073 strains of E. coli .
  • FIG. 12B shows the relative abundance of transcripts for the genes related to glucose utilization namely cyaA, crp, ptsG, galP and glk in ATCC8739, KJ012, KJ017, KJ060, KJ071 and KJ073 strains of E. coli .
  • FIG. 12C shows the relative abundance of transcripts for the genes ptsI and crr gene in the ATCC8739, KJ012, KJ017, KJ060, KJ071 and KJ073 strains of E. coli.
  • FIG. 13 Anaerobic metabolism of E. coli using the mixed acid fermentation pathway. (Bock & Sawers, 1996). The native mixed acid pathway is shown with black arrows. Additional reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown with dotted arrows. Dotted bold arrows indicate new metabolic steps that have been recruited for succinate production in E. coli mutants. Reactions that have been blocked by gene deletions or point mutations are marked with an X. The pck* indicates a novel mutation that de-repressed phosphoenolpyruvate carboxykinase, increasing activity and allowing this enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites in this diagram but exists as a single intracellular pool.
  • FIG. 14 Generally recognized pathways for the glycerol catabolism combined with mixed acid fermentation (anaerobic) in wild type E. coli . These pathways are based on a combination of the most current reviews in EcoSal, data available in Ecocyc, and a review of primary literature. (Bachler et al., 2005; Berman and Lin, 1971; Bock and Sawers, 1996; Erni et al., 2006; Gutknecht et al., 2001; Jin et al., 1983; Keseler et al., 2005; Lin, 1996; Tang et al., 1982). Bold arrows indicate the pathways generally regarded as dominant for glycerol catabolism and for mixed acid fermentation.
  • Thin arrows show a pathway regarded as cryptic and nonfunctional in wild type E. coli (Jin et al., 1983; Tang et al., 1982) involving dihydroxyacetone as an intermediate. This pathway is thought to function only in mutants in which glpK is inactive. (Jin et al., 1983; Tang et al., 1982).
  • a thin arrow is also shown for glpD, the dehydrogenase thought to function during aerobic metabolism as a replacement for glpABC (anaerobic metabolism).
  • DHA dihydroxyacetone
  • DHAP dihydroxyacetone 3-phosphate
  • PEP phosphoenolpyruvate
  • G3P glycerol 3-phosphate
  • GA3P glyceraldehydes 3-phosphate.
  • PEP is boxed to indicate a common pool.
  • FIG. 15 Novel E. coli pathway for the anaerobic production of succinate from glycerol in mineral salts medium.
  • Bold arrows indicate the primary route for the anaerobic catabolism of glycerol in strains such as XZ721 containing three core mutations for succinate production. Dotted arrows show pathways that have been blocked by mutations in pflB and ptsI.
  • mutational activation of phosphoenolpyruvate carboxykinase (pck*) allows this enzyme to serve as the dominant carboxylation step for the production of oxaloacetate.
  • phosphoenolpyruvate carboxylase PPC
  • PCK conserves energy to produce additional ATP for biosynthesis.
  • titer means the molar concentration of particular compound in the fermentation broth.
  • a succinic acid titer of 100 mM would mean that the fermentation broth at the time of measurement contained 100 mMoles of succinic acid per liter of the fermentation broth.
  • yield refers to the moles of particular compound produced per mole of the feedstock consumed during the fermentation process.
  • yield refers to the number of moles of succinic acid produced per mole of glucose consumed.
  • volumetric productivity refers to the amount of particular compound in grams produced per unit volume per unit time.
  • a volumetric productivity value of 0.9 g L ⁇ 1 h ⁇ 1 for succinic acid would mean that 0.9 gram succinic acid is accumulated in one liter of fermentation broth during an hour of growth.
  • titer “yield,” and “volumetric productivity” as used in this invention also include “normalized titer,” “normalized yield,” and “normalized volumetric productivity.”
  • normalized titer “normalized yield”
  • volumetric productivity the volume of the neutralizing reagents added to the fermentation vessel in order to maintain the pH of the growth medium is also taken into consideration.
  • genetically engineered or “genetically modified” as used herein refers to the practice of altering the expression of one or more enzymes in the microorganisms through manipulating the genomic DNA of the microorganisms.
  • the present invention provides a process for the production of succinic acid in commercially significant quantities from the carbon compounds by genetically modified bacterial strains (GMBS).
  • GMBS genetically modified bacterial strains
  • Disclosed in this present invention are the microorganisms suitable for the production of succinic acid through fermentative process.
  • the term “gene” includes the open reading frame of the gene as well as the upstream and downstream regulatory sequences.
  • the upstream regulatory region is also referred as the promoter region of the gene.
  • the downstream regulatory region is also referred as the terminator sequence region.
  • the term mutation refers to genetic modifications done to the gene including the open reading frame, upstream regulatory region and downstream regulatory region.
  • the gene mutations result either an up regulation or a down regulation or complete inhibition of the transcription of the open reading frame of the gene.
  • the gene mutations are achieved either by deleting the entire coding region of the gene or a portion of the coding nucleotide sequence or by introducing a frame shift mutation, a missense mutation, and insertion, or by introducing a stop codon or combinations thereof.
  • exogenous is intended to mean that a molecule or an activity derived from outside of a cell is introduced into the host microbial organism.
  • the introduced nucleic acid may exist as an independent plasmid or may get integrated into the host chromosomal DNA.
  • the exogenous nucleic acid coding for a protein may be introduced into the microbial cell in an expressible form with its own regulatory sequences such as promoter and terminator sequences.
  • the exogenous nucleic acid molecule may get integrated into the host chromosomal DNA and may be under the control of the host regulatory sequences.
  • exogenous refers to the molecules and activity that are present within the host cell.
  • exogenous refers to an activity that is introduced into the host reference organism.
  • the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. If the nucleic acid coding for a protein is obtained from the same species of the microbial organism, it is referred as homologous DNA. If the nucleic acid derived from a different microbial species, it is referred as heterologous DNA.
  • exogenous expression of an encoding nucleic acid of the invention can utilize either or both heterologous and homologous encoding nucleic acid.
  • the present invention provides GMBS showing impressive titers, high yield and significant volumetric productivity for succinic acid when grown under fermentative conditions in minimal salt medium containing a carbon source as the substrate for fermentation process.
  • the microorganisms of the subject invention can be employed in a single step production process using various sugars such as hexoses, pentoses, disaccharides and other carbon compounds such as glycerol.
  • succinic acid production is coupled with microbial growth which in turn is coupled to cellular ATP level and redox balance.
  • redox balance refers to the ability of the cell to maintain the appropriate ratio of NADH to NAD + .
  • the cells are able to oxidize the NADH so that there is enough NAD + to oxidize the carbohydrate substrates during the anaerobic fermentative growth.
  • the NAD + pool is regenerated through oxidative phosphorylation involving NADH.
  • the regeneration of NAD + pool is achieved only by means of manipulating the flow of carbon through various metabolic pathways inside the cell which could oxidize NADH.
  • the genetic modifications involve only the manipulation of genes within the native genome of the microorganisms.
  • no exogenous genetic material such as plasmid bearing antibiotic resistance genes or any other exogenous nucleotide sequences coding for certain enzyme proteins is introduced into the bacterial strains used as a biocatalysts for succinic acid production.
  • the recombinant microorganisms suitable for this present invention are derived from a number of bacterial families, preferably from the Enterobacteriaceae family.
  • the suitable microorganisms are selected form the genera Escherichia, Erwinia, Providencia , and Serratia .
  • the genus Escherichia is particularly preferred. Within the genus Escherichia , the species Escherichia coli is particularly preferred. Any one strain of E. coli such as E. coli B, E. coli C, E. coli W, or the like is useful for the present invention.
  • E. coli strains capable of producing organic acids in significant quantities are well known in the art.
  • the U.S. Patent Application Publication No. 2009/0148914 provides strains of E. coli as a biocatalyst for the production of chemically pure acetate and/or pyruvate.
  • the U.S. Patent Application Publication No. 2007/0037265 and U.S. Pat. No. 7,629,162 provide derivatives of E. coli K011 strain constructed for the production of lactic acid.
  • International Patent Application published under the Patent Cooperation Treaty No. WO 2008/115958 provides microorganism engineered to produce succinate and malate in minimal mineral salt medium containing glucose as a source of carbon in pH-controlled batch fermentation.
  • bacteria that can be modified according to the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticurn, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricaturn, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium testaceum, Bre
  • the microorganisms suitable for the practice of present invention can be grown aerobically (in the presence of oxygen) or anaerobically (in the complete absence of oxygen) or micro aerobically (with a minimal amount of oxygen supply).
  • the microorganism selected for the production of succinic acid is grown in an anaerobic condition.
  • the microorganisms suitable for the present invention can be grown in a dual-phase growth regime, wherein the microorganism is initially grown in aerobic growth condition to reach a certain level of cell growth before transferring it to the anaerobic growth condition to achieve the production succinic acid in commercially significant quantities.
  • production and the accumulation of the succinic acid occurs during the anaerobic fermentative growth phase.
  • the present invention combines the technique of specific genetic modifications with the process of metabolic evolution to obtain strains showing high yield, titer and volumetric productivity for succinic acid production under anaerobic growth condition in the mineral salt medium with a carbohydrate substrate.
  • the microbial strains obtained from genetic manipulations would have the expected genotype for the production of succinic acids. However, their growth rate in the minimal mineral salt medium or the their ability to produce succinic acid at the required yield, titer and volumetric productivity may not allow us to use these genetically modified microorganism as a biocatalyst for the commercial production of succinic acid through large scale fermentation process. Therefore, the genetically modified microbial strains obtained from genetic modifications are subsequently grown in mineral salt medium with a carbohydrate source for several generations to select a clone with very high yield for succinic acid production. This process for the growth-based selection of a clone with most preferred phenotype is referred as metabolic evolution.
  • the genetically modified strain is repeatedly transferred into fresh minimal medium for a period of time to obtain a clone in which the spontaneous mutations that occurred during metabolic evolution results in a clone that exhibits fast cell growth, rapid consumption of different carbon sources, ability to use multiple sugars simultaneously, ability to tolerate toxic chemicals in the carbon source and high production yield and productivity of the desired organic acid coupled with the low production of other organic acids.
  • a clone resulting from the metabolic evolution showing a very good growth rate in mineral medium supplemented with a carbon source but that has not improved in the yield of the desired organic acid is not a desirable clone.
  • the organism could simply adapt itself to the selective pressure and show a changed phenotype.
  • the organism might undergo certain genetic changes under selective pressure and exhibit a changed phenotype permanently.
  • the organism reverts back to its original phenotype once the selection pressure is relieved.
  • These organisms are referred to as “adapted” organisms.
  • These “adapted” microorganisms would revert back to their original phenotype when the selection pressure is removed.
  • the “adapted” microorganisms have to undergo another fresh round of metabolic evolution under selection pressure to show a changed phenotype.
  • Metabolic evolution accompanied by a certain genetic change is desirable.
  • the microorganism acquiring a stable genetic change during metabolic evolution can be easily identified by means of growing the microorganism in the original growth medium without any selection pressure for some time before transferring it to the fresh medium with the selection pressure. If these organisms are able to show good growth and the expected phenotype without any lag period, the organism is considered to have acquired a changed genotype during metabolic evolution.
  • the basis of genetic change gained during the metabolic evolution can be determined by sequencing appropriate regions within the chromosomal DNA of the organism and comparing the sequence data with that of the parent strain.
  • the DNA sequence data can be obtained by means of following the techniques well known in the art. For example, appropriate regions of the chromosomal DNA of the metabolically evolved strain can be obtained through polymerase chain reaction and the product obtained through polymerase chain reaction can be sequenced by using appropriate sequencing primers.
  • the wild type E. coli strains obtained from culture collections such as ATCC can be genetically engineered and subsequently metabolically evolved to obtain a strain with an enhanced ability to produce succinic acid in commercially significant amounts.
  • the genetic manipulations can be done in several different stages accompanied by metabolic evolution in between the stages of genetic manipulations.
  • the genomic manipulations involve either altering the endogenous DNA sequences or completely removing specific DNA sequences from the genomic DNA.
  • the genetic manipulations may also involve inserting a foreign DNA sequence within the genomic DNA sequence of the microorganism.
  • the genetic manipulations are accomplished by means of removing specific DNA sequences from the genomic DNA of the microorganisms without introducing any foreign DNA.
  • Certain genetic manipulations necessary to inactivate the expression of a gene coding for a particular protein product requires an insertion of a foreign DNA sequence into the genome of the microorganism to select a clone with the desired genetic modification.
  • exogenous antibiotic marker genes can be used to insertionally inactivate the endogenous genes and to select the clone with desired genotype.
  • the introduced exogenous DNA sequences are ultimately removed from the genomic DNA of the microorganism so that the microorganism at the end of the genetic engineering process would have no exogenous DNA in its original genomic DNA.
  • Patent Applications with numbers US 2007/0037265 and US 2009/0148914 and the International patent application published under the Patent Cooperation Treaty with International Publication Number WO 2008/115958 also describe the genetic engineering techniques useful in practicing various embodiments of this present invention.
  • These scientific publications as well as patent documents are herein incorporated by reference for the purpose of providing the details for genetic engineering techniques useful for the present invention.
  • the microbial carbon metabolism involves glycolysis, tricarboxylic acid cycle and oxidative phosphorylation.
  • the reduced enzyme co-factors such as NADPH and NADH are regenerated by the operation of oxidative phosphorylation accompanied by ATP production required for cell growth.
  • the regeneration of reduced cofactors NADPH and NADH is accomplished by directing the carbon flow into the tricarboxylic acid cycle and eliminating all of the fermentative pathways for regeneration of NADP ⁇ and NAD + .
  • the metabolic pathways are specifically engineered so that the microorganism produces a particular organic acid of our choice.
  • the microorganisms are capable of synthesizing a number of organic acids including lactic acid, acetic acid, malic acid, pyruvic acid, formic acid and succinic acid.
  • succinic acid the pathways for production of acetic acid, lactic acid, pyruvic acid, and formic acid are blocked and the carbon flow to succinic acid production is facilitated through manipulating one or more enzymes involved in the carbon metabolism within the cell.
  • the list of the enzymes that are active in the microbial fermentative pathway which can be manipulated using the known genetic engineering techniques includes, but not limited to, isocitrate synthetase (aceA), malate synthase (aceB), the glyoxylate shunt operon (aceBAK), acetate kinase-phosphotransacetylase (ackA-pta); aconitase hydratase 1 and 2 (acnA and acnB); acetyl-CoA synthetase (acs); citrate lyase (citDEF); alcohol dehydrogenase (adhE); citrate synthase (citZ); fumarate reductase (frd); lactate dehydrogenases (ldh); malate dehydrogenase (mdh); aceBAK operon repressor (iclR); phosphoenol pyruvate carboxylase (pepC);
  • PEP phosphoenol pyruvate
  • mixed acid pathway refers to the flow of carbon from PEP through both tricarboxylic acid cycle and various fermentative pathways that are operational under anaerobic conditions. Under anaerobic conditions, at least four different fermentative pathways for the metabolism of pyruvate are recognizable.
  • the pyruvate may be reduced to lactate using the NADH and thereby producing NAD + to maintain the redox balance of the cell necessary for the continuous metabolism of carbon source.
  • the acetyl-CoA derived from pyruvate may also be reduced to produce ethanol accompanied by the oxidation of NADH to produce NAD + .
  • Pyruvate may also be converted into formate or acetate as shown in FIG. 1A .
  • the oxidative min encompassing the carbon flow from oxaloacetate to succinic acid through isocitrate
  • the NADP + is utilized to oxidize isocitrate with the resulting formation of NADPH.
  • the reductive arm of the TCA cycle encompassing the flow of carbon from oxaloacetate to succinic acid through malate and fumarate
  • the NADH is oxidized to produce NAD + and thereby helping the cell to maintain the redox balance.
  • the carbon flow from PEP through fermentative pathways is prevented by mean of inactivating the genes coding for the enzymes involved in the fermentative pathway.
  • the enzymes suitable for blocking the carbon flow through fermentative pathway include ldhA, pflB, adhE, pta, ackA, and poxB. The elimination of one or more of these genes is expected to reduce the carbon flow from PEP through the fermentative pathway. Inactivation of all these six genes is expected to block the carbon flow through fermentative pathway totally.
  • the mgsA gene coding for the methylglyoxal synthase (mgsA) responsible for the conversion of methylglyoxal to lactic acid is inactivated beside the inactivation of six other genes involved in the fermentative pathway.
  • the functional homologues of the genes involved in the fermentative pathway are also inactivated besides inactivating the genes well known to be involved in one or other fermentative pathway.
  • a propionate kinase with acetate kinase activity is encoded by the tdcD gene which is produced only for the degradation of threonine.
  • the expression of tdcD could functionally replace ackA.
  • the adjacent tdcE gene in the same operon is similar to pflB and encodes ⁇ -ketobutryate formate lyase with pyruvate formate-lyase activity.
  • the tdcDE genes are inactivated to prevent the entry of carbon into fermentative pathway and to assure the flow of carbon into the TCA cycle.
  • the carbon flow within the TCA cycle is altered so that there is carbon flow directed towards the production of succinic acid.
  • the manipulation of carbon flow within the TCA cycle is achieved by means of up-regulating the expression of one or more genes.
  • one or more genes functioning within TCA cycle may be inactivated to facilitate an increased carbon flow to succinic acid.
  • the gene mdh encoding for malate dehydrogenase is up regulated to improve the conversion of malate to fumarate and succinate.
  • the flow of the carbon from oxaloacetate to succinic acid through malate and fumarate is referred as the reductive arm of the TCA cycle.
  • the flow of carbon through this reductive arm of the TCA cycle from oxaloacetic acid to succinic acid would consume two moles of NADH for every mole of succinic acid produced and thereby help in maintaining the redox balance of the cell under anaerobic condition.
  • the up-regulation of mdh would help in regenerating the NAD + required to maintain the redox balance of the cell.
  • the up-regulation of mdh gene expression can be achieved by means of replacing the native promoter for mdh gene with some other strong promoter sequence or alternatively by means of mutating the promoter region of the mdh gene so that there is an increase transcription of mdh gene. Alternatively, additional copies of the mdh gene can be added to the strain. In a preferred embodiment of the present invention, the up regulation of mdh gene expression is achieved by means of genetically manipulating its promoter region.
  • succinic acid is produced through the operation of oxidative of arm of the TCA cycle.
  • the flow of carbon from oxaloacetate to succinic acid through citrate, cis-aconitate, isocitrate, ⁇ -ketoglutarate, and succinyl-CoA is referred as the oxidative arm of the TCA cycle.
  • the succinic acid can also be produced through the operation of glyoxylate bypass. During the operation of glyoxylate bypass, by the action of isocitrate lyase, succinate and glyoxylate are produced from isocitrate.
  • succinate dehydrogenase succinate dehydrogenase
  • the carbon flow through the glyoxylate bypass can be manipulated to achieve an increase in the succinic acid production.
  • Isocitrate lyase enzyme catalyzes the cleavage of isocitrate to glyoxylate and succinate.
  • Isocitrate lyase is coded by the aceBAK operon.
  • the isocitrate lyase activity is suppressed by iclR genes.
  • the expression of iclR gene prevents the operation of glyoxylate shunt.
  • the iclR gene is inactivated beside the inactivation of the genes involved in the fermentative metabolism.
  • the outward carbon flow from the TCA cycle to other metabolic pathways can also be blocked through genetic means to increase the succinic acid production.
  • the flow of carbon from the TCA cycle into amino acid metabolism can be blocked in order to improve the carbon flow towards succinic acid.
  • the aspartate aminotransferase gene (aspC) transfers the amino group from glutamic acid to oxaloacetic acid in the synthesis of aspartic acid and thereby facilitates the outward flow of carbon from the TCA cycle.
  • the inactivation of the aspC gene is followed to block the outward flow of carbon from the TCA cycle in order to improve the carbon flow from oxaloacetate towards succinic acid production either through the oxidative or reductive arm of the TCA cycle.
  • the other outward flow of the carbon from TCA cycle occurs from malate.
  • the decarboxylation of malate by malic enzyme (sfcA) results in the production of pyruvate.
  • the gene coding for the sfcA gene is inactivated to curtail the outward flow of carbon from TCA cycle.
  • both aspC and sfcA genes are inactivated to prevent the outward flow of carbon from TCA cycle so as to enhance the succinic acid accumulation.
  • the outward flow of carbon from TCA cycle is prevented by inactivating the citrate lyase gene (citDEF) responsible for the cleavage of citric acid into oxaloacetate and acetate.
  • citDEF citrate lyase gene
  • the present invention has also surprisingly discovered that the growth coupled succinic acid yield can further be improved by genetic manipulations of carboxylating enzymes within the microbial cells. While characterizing the changes that occurred during the metabolic evolution through conducting genetic and enzyme analysis, the inventors of the present invention have unexpectedly discovered that the carboxylating enzymes within the cell could be yet another target for genetic manipulation to achieve an improved succinic acid yield.
  • the glycolytic intermediates phosphoenol pyruvate (PEP) and pyruvic acid can be carboxylated to improve the carbon flow into the TCA cycle.
  • the carbon entry into the TCA cycle is accomplished by the action of citrate synthase which combines the acetyl-CoA derived from pyruvate with oxaloacetate, an intermediate in TCA cycle, to produce citric acid.
  • citrate synthase which combines the acetyl-CoA derived from pyruvate with oxaloacetate, an intermediate in TCA cycle, to produce citric acid.
  • the present invention provides a method for manipulating the carboxylating enzymes present within the cell as a method to increase the succinic acid yield during anaerobic fermentative growth. It is well known in the art that by means of introducing pyruvate carboxylase (pyc) from an exogenous source it is possible to carboxylate pyruvate to oxaloacetic acid.
  • pyc pyruvate carboxylase
  • the microbial strains well suited for genetic manipulations such as E. coli do not have the pyc gene.
  • the pyc genes derived from other bacterial species such as Rhizopium elti and Lactobacillus lacti can be introduced into the genetically modified E. coli strains to improve succinic acid production.
  • carboxylating enzymes Four different endogenous carboxylating enzymes are known in E. coli . Two of these enzymes are responsible for carboxylating phosphoenol pyruvate and two other enzymes are responsible for the carboxylation of pyruvate derived from phosphoenol pyruvate by the action of pyruvate kinase enzyme.
  • the enzyme phosphoenol pyruvate carboxylase (ppc) carboxylates phosphoenol pyruvate to oxaloacetate which could enter into reductive arm of the TCA cycle to produce succinate.
  • the second carboxylating enzyme phosphoenol pyruvate carboxykinase also carboxylates phosphoenol pyruvate to produce oxaloacetate, but normally catalyzes the reverse reaction as it is not expressed in the presence of glucose.
  • the two other carboxylating enzymes namely NADH-linked maleic enzyme (maeB) and the NADPH-linked maleic enzyme (maeA/sfcA) carboxylate pyruvic acid to malic acid.
  • the maeB and sfcA enzymes carboxylates the pyruvate derived from phosphoenol pyruvate by the action of pyruvate kinase.
  • any one of the four carboxylating enzymes present in the cell can be genetically manipulated to increase its enzymatic activity in order to improve the carbon flow from glycolytic cycle intermediates into the TCA cycle.
  • the PPC-catalyzed reaction is strongly favored. Energy contained in PEP is lost in this reaction with the release of inorganic phosphate.
  • the other three carboxylating enzymes namely pck, maeA and sfcA (maeB), are not expected to function during the fermentative growth using glucose as the substrate as these three carboxylating enzymes are repressed by glucose. These three carboxylating enzymes are thought to function in the reverse direction during gluconeogenesis when the cells are oxidatively metabolizing organic acids.
  • gluconeogenic PEP carboxykinase can be genetically manipulated to improve the flow of carbon into the TCA cycle.
  • PCK gluconeogenic PEP carboxykinase
  • the comparative analysis of phosphoenol pyruvate carboxykinase activity in a number of E. coli strains constructed during this invention has revealed that the increase in the PCK enzyme activity during the metabolic evolution results from an increase in the transcriptional activity of the pck gene. In those strains showing an improvement in the cell growth-coupled succinate production, there is an increase in the abundance of the pck transcript. In fact, the results of the present invention have established a positive correlation between an increase in the pck transcript level and an increase in the PCK enzyme activity and growth-coupled succinic acid production.
  • the recruitment of the native gluconeogenic pck for fermentative succinate production can be achieved by any mutation that positively affects the transcription of the pck gene.
  • An increase in the level of PCK activity can be achieved by means of expressing the pck gene in a multicopy plasmid with a native promoter or any other promoter sequence which is known to increase the gene's expression.
  • Another way to increase the expression of the pck gene within the cell is to integrate additional copies of the pck gene using transposons.
  • the native promoter of the pck gene can be replaced by some other promoter elements known to enhance the level of activity.
  • An increased expression of pck gene can also be achieved either by mutation in the promoter region of the gene or by genetic manipulation of the regulatory elements that are known to interact with the promoter region of the pck gene.
  • the gene coding for a regulator protein of the pck gene can be mutated or deleted or overexpressed in some way in order to increase the expression of pck gene.
  • the results of the present invention have indicated that a single point mutation (G to A transition at position—64 relative to the ATG start codon of pck gene) could increase the transcription of the pck gene accompanied by a corresponding increase in the phosphoenol pyruvate carboxykinase enzyme activity.
  • a similar increase in the pck gene expression can also achieved by genetically manipulating the genes coding for the proteins known to regulate the expression of pck gene.
  • Cra protein has been shown to activate the expression of pck gene in E. coli (Saier and Ramseier, 196).
  • the csrA system (comprising csrA, csrB, csrC, csrD, uvrY or barA) has also been reported to regulate the level of pck and other genes involved in glucose metabolism by altering mRNA stability (Babitzke and Romeo, 2007; Pernestig et al., 2003; Suzuki K et al., 2002).
  • Yet another genetic approach of the present invention to increase the growth-coupled succinic acid production during the anaerobic fermentation process is concerned with the conservation of energy expended in sugar uptake by the biocatalysts.
  • the microorganisms take up the sugars through a set of transporter proteins located on the cytoplasmic membrane (Jojima et al., 2010).
  • the microbial sugar transporters fall within three major categories.
  • the largest group of sugar transporters in the bacteria is known as ATP binding cassette (ABC) transporters.
  • ABC transporters require a molecule of ATP for every molecule of sugar transported into the bacterial cell.
  • XylFGH is an ABC transporter for the transport of xylose, a pentose sugar, into the cell.
  • AraFGH is an ABC transporter for the transport of arabinose, yet another pentose sugar.
  • MFS Facilitator Super family
  • H + -linked symporters H + -linked symporters
  • Na + -linked symporters-antiporters H + -linked symporters
  • uniporters simple facilitators for the sugar transport and require a molecule of ATP for every molecule of sugar transported into the cell.
  • the trans-membrane protein Glf in E. coli is an example of uniporter.
  • the H + -symporters require a proton and a molecule of ATP for every sugar molecule transported into the cell.
  • GalP galactose
  • GalP a very well characterized symporter with 12 trans-membrane loops. GalP is also reported to have the ability to transport glucose across the cell membrane.
  • AraE is a proton-linked symporter for the transport of arabinose across the cell membrane.
  • XylE protein is a proton-linked symporter for the transport of xylose.
  • the third sugar transporter primarily responsible for the uptake of hexose sugars such as glucose is known as the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS).
  • PTS carbohydrate phosphotransferase system
  • the phosphotransferase system genes ptsH and ptsG can be manipulated to conserve the energy in glucose uptake and thereby improve the efficiency of succinic acid production by microorganism. Thus by mining the data available in the area of microbial metabolic pathways, one can delete a set of genes so as to block most of the metabolic pathways and channel the carbon flow to the production of succinic acid with great efficiency.
  • PTS is comprised of two cytoplasmic components namely EI and HPr and a membrane-bound component EII.
  • E. coli contains at least 15 different EII complexes.
  • Each EII component is specific to a sugar type to be transported and contains two hydrophobic integral membrane domains (C and D) and two hydrophilic domains (A and B). These four domains together are responsible for the transport and phosphorylation of the sugar molecules.
  • EI protein transfers the phosphate group from PEP to HPr protein.
  • EII protein transfers the phosphate group from phosphorylated HPr protein to the sugar molecule.
  • EI is encoded by the ptsI gene.
  • HPr is encoded by the ptsH gene.
  • the glucose-specific EII complex of enteric bacteria consists of two distinct proteins namely, EIIA Glc encoded by the gene crr and the membrane-associated protein EIICB Glc encoded by the gene ptsG.
  • the PTS mediated sugar transport can be inhibited by means of deleting one of these genes coding for the proteins associated with PTS.
  • Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities is one of the genetic approaches for achieving significant improvements in product yield from glucose and productivity for several classes of metabolites.
  • GalP has been reported to transport glucose in the pts ⁇ strain
  • the significance of GalP mediated glucose uptake is evidence by the fact that the inactivation of galP gene in the pts ⁇ mutant is found to be lethal (Yi et al., 2003).
  • Glk is necessary to achieve the phosphorylation of the glucose molecule before it can enter into glycolysis.
  • the expression of the GalP protein in the pts ⁇ strain can be achieved either by expressing an exogenous gene under a constitutive promoter or by means of relieving the repression of the galP expression through mutations in genes coding for the repressor of the galP gene such as galS and galR.
  • the succinic acid production using microbial catalysts can be achieved by means of genetic manipulation accompanied by metabolic evolution.
  • the genetic changes that occur during the metabolic evolution can be identified through biochemical and genetic analysis.
  • the present invention have surprisingly discovered that mutations occurring in the genes for the phosphotransferase system and carboxylating enzymes present within the cell could positively contribute to the increase in the succinic acid production.
  • These newly discovered targets for genetic manipulations can be combined with the other targets in the glycolytic, tricarboxylic acid and fermentative pathways in several different ways to generate biocatalysts with high efficiency for succinic acid production. It is also highly desirable to identify a set of a minimal number of target genes for genetic manipulation in order to achieve the best succinic acid producing strains.
  • the invention also provides genetic approaches to enhance glycerol utilization in succinic acid production.
  • the glycerol uptake is mediated by the protein coded by glpF gene. Once taken into the cell, glycerol is oxidized by the protein coded by gldA gene to produce dihydroxy acetone (DHA).
  • DHA dihydroxy acetone
  • DHAP dihydroxy acetone phosphate
  • the phosphorylation of DHA to DHAP by the proteins coded by dhaKLM is dependent on the availability of the phosphoenol pyruvate (PEP) pool.
  • the present invention has surprisingly found that preventing the flow of glycerol through the gldA and dhaKLM pathways in a bacterial cell having an increased PCK enzymatic activity could enhance the succinic acid yield using glycerol as the carbon source.
  • the present invention has also surprisingly discovered that further improvement in succinic acid yield using glycerol as the carbon source can be achieved by means of preventing the carbon flow through fermentative pathways in a bacterial cell with improved PCK enzyme activity and deletions in the gldA gene and dhaKLM operon.
  • E. coli C ATCC 8739
  • ATCC 8739 New derivatives of E. coli C
  • the various strain of E. coli developed in the present invention have been deposited with ARS Culture Collection with accession numbers as shown in Table 2.
  • the microbial organism of the present invention can be grown in a number of different culture media well known in the field of microbiology.
  • the wild type and mutant strains of E. coli are grown in Luria-Bertani (LB) medium containing 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl.
  • LB Luria-Bertani
  • a minimal mineral salt medium supplemented with a carbon source is preferred.
  • the use of a minimal mineral salt medium as opposed to a rich medium like LB medium reduces the cost for the production of organic acids in a commercial scale.
  • the minimal mineral mediums suitable for the present invention include NBS medium (Causey et al., 2007) and AMI medium (Martinez et al., 2007).
  • the NBS medium contains 1 mM betaine, 25.72 mM KH 2 PO 4 , 28.71 mM K 2 HPO 4 , 26.50 mM (NH 4 )2HPO 4 , 1 mM MgSO 4 .7H2O, 0.1 mM CaCl 2 .2H 2 0, 0.15 mM Thiamine HCl, 5.92 ⁇ M FeCl 3 6H 2 O, 0.84 COCl 2 .6H 2 O, 0.59 ⁇ M CuCl 2 .2H 2 O, 1.47 ⁇ M ZnCl 2 , 0.83 ⁇ M Na 2 MoO 4 2H 2 O, and 0.81 ⁇ M H 3 BO 3 .
  • the AM1 medium contains 1 mM betaine, 19.92 mM (NH 4 )2HPO 4 , 7.56 mM NH 4 H 2 PO 4 , 1.5 mM MgSO 4 .7H2O, 1.0 mM Betaine-KCl, 8.88 ⁇ M FeCl 3 6H 2 0, 1, 26 ⁇ M COCl 2 .6H 2 O, 0.88 ⁇ M CuCl 2 .2H 2 O, 2.20 ⁇ M ZnCl 2 , 1.24 ⁇ M Na 2 MoO 4 2H 2 O, 1.21 ⁇ M H 3 BO 3 and 2,50 ⁇ M MnCl 2 4H 2 O.
  • the trace elements are prepared as a 1000 ⁇ stock and contained the following components: 1.6 g/L FeCl 3 , 0.2 g/L CoCl 2 .6H 2 O, 0.1 g/L CuCl 2 , 0.2 g/L ZnCl 2 .4H 2 O, 0.2 g/L NaMoO 4 , 0.05 g/L H 3 BO 3 , and 0.33 g/L MnCl 2 .4H 2 O.
  • the mineral medium for microbial production of organic acid is supplemented with a carbon source.
  • the carbon sources useful in the present invention include but are not limited to pentose sugars like xylose, hexose sugars like glucose, fructose, and galactose and glycerol.
  • the carbon source can also be satisfied by providing a combination of different sugars such as a combination of glucose and xylose.
  • the carbon source can also be derived from a hydrolysis of starch or lignocellulose.
  • the hydrolysis of complex carbohydrates such as starch and lignocelluloses can be achieved either by using thermo-chemical conversion processes or enzymatic methods well known in the art.
  • the preferred carbon source for the industrial production of organic acid using microbial fermentation is lignocellulosic hydrolysate derived from the hydrolysis of agricultural or forestry wastes.
  • the lignocellulosic hydrolysate may further be fractionated to yield a hexose-enriched and a pentose-enriched fraction and those fractions can serve as the source of carbon for the commercial production of the organic acids using microbial fermentation process.
  • the lignocellulosic hydrolysate can further be detoxified to remove certain chemicals such as furfural which are found to be toxic to a number of microbial organisms above certain concentrations.
  • Bacterial strains, plasmids, and primers used in this study are listed in Tables 3, 7, 9, 14 and 15 and are explained in detail at the appropriate places in the specification below.
  • Luria broth (10 g l ⁇ 1 Difco tryptone, 5 g l ⁇ 1 Difco yeast extract and 5 g l ⁇ 1 NaCl) containing 2% (w/v) glucose or 5% (w/v) arabinose.
  • No genes encoding antibiotic resistance, plasmids, or foreign genes are present in the final strains developed for succinate production.
  • various antibiotic resistance markers were used.
  • the antibiotics such as ampicillin (50 mg l ⁇ 1 ), kanamycin (50 mg l ⁇ 1 ), or chloramphenicol (40 mg l 1 ) were added as needed for antibiotic selection process.
  • Seed cultures and fermentations were grown at 37° C., 100 rpm in NBS or AM1 mineral salts medium containing glucose, 100 mM KHCO 3 and 1 mM betaine HCl.
  • corn steep liquor was used. It is a byproduct from the corn wet-milling industry. When compared to the yeast extract and peptone, it is an inexpensive source of vitamins and trace elements.
  • strains were grown without antibiotics at 37° C. in NBS mineral salts medium (Causey et al., 2004) supplemented with 10% (w/v) glucose and 100 mM potassium bicarbonate unless stated otherwise.
  • Pre-inocula for fermentation were grown by transferring fresh colonies into a 250 ml flask (100 ml NBS medium, 2% glucose). After 16 h (37° C., 120 rpm), this culture was diluted into a small fermentation vessel containing 300 ml NBS medium (10% glucose, 100 mM potassium bicarbonate) to provide an inoculum of 0.033 g cell dry wt (CDW) l ⁇ 1 .
  • CDW cell dry wt
  • the pH of the culture vessel can be continuously monitored using a pH probe, and appropriate base can be added to maintain the pH of the growth medium around neutral pH.
  • the bases suitable for maintaining the pH of the microbial culture include, but not limited to, NaOH, KOH, NH 4 SO 4 , Na 2 CO 3 , NaHCO 3 , and NH 4 CO 3 .
  • the bases suitable for this purpose can be used alone or in combination.
  • fermentations were automatically maintained at pH 7.0 by adding base containing additional CO 2 (2.4 M potassium carbonate in 1.2 M potassium hydroxide). Subsequently, pH was maintained by adding a 1:1 mixture of 3M K 2 CO 3 and 6N KOH. Fermentation vessels were sealed except for a 16 gauge needle which served as a vent for sample removal. Anaerobiosis was rapidly achieved during growth with added bicarbonate serving to ensure an atmosphere of CO 2 .
  • chromosomal genes were deleted seamlessly without leaving segments of foreign DNA as described previously. (Jantama et al., 2008a, 2008b; Zhang et al., 2007). Red recombinase technology (Gene Bridges GmbH, Dresden, Germany) was used to facilitate chromosomal integration. Plasmids and primers used during construction are listed in Tables 3, 7, 9, 14, and 15. Plasmids and primers used in the construction of the strains KJ012, KJ017, KJ032, KJ060, KJ070, KJ071, KJ072, KJ073, and SZ204 are summarized in Table 3.
  • Sense primers contain sequences corresponding to the N-terminus of each targeted gene (boldface type) followed by 20 bp (underlined) corresponding to the FRT-kan-FRT cassette.
  • Anti-sense primers contain sequences corresponding to the C-terminal region of each targeted gene (boldface type) followed by 20 bp (underlined) corresponding to the cassette.
  • Amplified DNA fragments were electroporated into E. coli strains harboring Red recombinase (pKD46). In resulting recombinants, the FRT-kan-FRT cassette replaced the deleted region of the target gene by homologous recombination (double-crossover event).
  • the resistance gene (FRT-kan-FRT) was subsequently excised from the chromosome with FLP recombinase using plasmid pFT-A, leaving a scar region containing one FRT site. Chromosomal deletions and integrations were verified by testing for antibiotic markers, PCR analysis, and analysis of fermentation products.
  • Generalized P1 phage transduction (Miller, 1992) was used to transfer the ⁇ focA-pflB::FRT-kan-FRT mutation from strain SZ204 into strain KJ017 to produce KJ032.
  • Plasmid pLOI4151 was used as a source of a cat-sacB cassette and Red recombinase (pKD46) was used to facilitate double-crossover, homologous recombination events. Chloramphenicol resistance was used to select for integration. Growth with sucrose was used to select for loss of sacB. With this approach, successive deletions were constructed to produce derivatives of KJ079 that eliminated all FRT sites. Primers and plasmids used in the removal of FRT markers from the adhE, ldhA and focA-pflB loci are listed in Table 3.
  • hybrid primers for ⁇ adhE::FRT target region were designed to contain approximately 50 bp of homology to the 5′ and 3′ regions of ⁇ adhE:: FRT site and 20 bp corresponding to cat-sacB gene from pLOI4151. These primers were used for PCR amplification of the cat-sacB cassette using pLOI4151 as a template.
  • the resulting PCR product was used to replace the FRT site in ⁇ adhE region with a cat-sacB cassette by a double-crossover, homologous recombination event with selection for resistance to chloramphenicol, to produce TG200.
  • the adhE gene and surrounding sequence were amplified from E. coli C using up/downadhE primers.
  • the PCR product containing chE′-adhE-ychG′ (3.44 kb) was cloned into pCR2.1-TOPO, yielding pLOI4413.
  • a second set of primers (IO-adhEup/down) was used to amplify the inside-out product with pLOI4413 as a template and Pfu polymerase to yield a blunt-ended product in which a 2.6 kb internal segment of adhE sequence was deleted.
  • This inside-out PCR product was kinase-treated and self-ligated, resulting in pLOI4419.
  • telomere sequence was replaced in TG200 with the desired chromosomal sequence by another double, homologous recombination event, with sucrose selection for loss of sacB.
  • the resulting strain was designated TG201 (KJ079 with the FRT removed from ⁇ adhE region).
  • the FRT sites in the ⁇ ldhA and ⁇ (focA-pflB) regions were removed in a manner analogous to that used to delete the adhE::FRT site. Additional primer sets (ldhAA/C and IO-ldhAup/down) used to remove the FRT site in ⁇ ldhA are included in Table 7 together with the corresponding plasmids (pLOI4430 and pLOI4432).
  • Strain TG202 was produced by replacing this region in TG201 with the PCR product from pLOI4151 (WMldhAA/C primers).
  • the cat-sacB cassette in TG202 was replaced with the PCR product from pLOI4432 (ldhAA/C primers) with sucrose selection for loss of sacB to produce TG203.
  • Primer sets (upfocA/MidpflA and IO-ycaOup/IO-midpflAdown) and corresponding plasmids (pLOI4415 and pLOI4421) used to remove the FRT site in ⁇ (focA-pflB) are included in Table 7.
  • Strain TG204 was produced by replacing this region in TG203 with the PCR product from pLOI4151 (WMpflBA/C primers).
  • the cat-sacB cassette in TG204 was replaced with the PCR product from pLOI4421 (upfocA/MidpflA primers) with sucrose selection for loss of sacB to produce KJ091.
  • KJ091 is a derivative of KJ073 in which all FRT sites have been removed from the ⁇ adhE, ⁇ ldhA and ⁇ focA-pflB regions of the chromosome.
  • plasmids containing sequences of the desired mutation were constructed as follows. E. coli C genomic DNA was used as the template for PCR amplification of ackA with the JMackAF1/R1 primers that bind approximately 200 bp upstream and downstream of the ackA gene. The linear product was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) to produce pLOI4158. Plasmid pLOI4158 was then used as a template for inside-out PCR with JMackAup1/down1 primers and Pfu polymerase to yield a blunt-ended product that lacks an 808-bp internal segment of ackA.
  • the PacI-flanked cat-sacB cassette (SmaI/SfoI fragment from pLOI4162) was then ligated into the blunt PCR product to produce pLOI4159.
  • Plasmid pLOI4159 served as a template for PCR amplification (JMackAF1/R1 primers). This PCR product was used to replace the FRT site in the ackA region of KJ073 by double-crossover homologous recombination, with selection for chloramphenicol resistance. The resulting clone was designated KJ076.
  • Plasmid pLOI4159 was also digested with PacI to remove the cat-sacB cassette and self-ligated to produce pLOI4160, retaining the 18-bp translational stop sequence.
  • Plasmid pLOI4160 served as a PCR template (JMackAF1/R1 primers). This amplified fragment was used to replace the cat-sacB cassette in KJ076 by double-crossover homologous recombination with selection for loss of sacB. After removal of pKD46 by growth at elevated temperature, the resulting strain was designated KJ079. In this strain, the deleted region has been replaced by the 18-bp translational stop sequence.
  • the strategy used above to remove the FRT site from the ackA region was employed to make sequential deletions of citF, sfcA and pta-ackA and to replace the deleted regions with the 18-bp translational stop sequence. Additional primer sets (citFup/down and citF2/3) used to construct the citF deletion are included in Table 7 together with the corresponding plasmids (pLOI4629, pLOI4630, and pLOI4631). The resulting strain was designated KJ104.
  • the sfcA gene was deleted from strains KJ104 and KJ110, resulting in strains designated KJ119 and KJ122, respectively. Additional primer sets (sfcAup/down and sfcA1/2) used to construct the sfcA deletions are included in Table 7 together with the corresponding plasmids (pLOI4283, pLOI4284, and pLOI4285).
  • the ackA-pta operon (including the synthetic translational stop sequence) was deleted from KJ122 to produce strain 10134. Additional primer sets (ackAup/ptadown and ackA2/pta2) used to construct this deletion are included in Table 7 together with the corresponding plasmids (pLOI4710, pLOI4711, and pLOI4712). Strain KJ134 does not contain any FRT sites or foreign genes.
  • plasmid pLOI4162 ( FIG. 8 ) was constructed with a removable cat-sacB cassette and the option to include an 8-bp segment of synthetic DNA with stop codons in all reading frames.
  • This plasmid is composed of synthetic sequences and parts of plasmids pLOI2228 (Martinez-Morales et al., 1999), pLOI2511 (Underwood et al., 2002), and pEL04 (Lee et al., 2001; Thomason et al, 2005).
  • Plasmid pLOI4131 was constructed by ligation of the FRT-cat-FRT fragment (Klenow-treated BanI, ClaI) from pLOI2228 into compatible sites of pLOI2511 (Klenow-treated NheI, ClaI).
  • Plasmid pLOI4131 was subsequently digested with EcoRI and self-ligated to remove the FRT-cat-FRT fragment to produce pLOI4145, retaining single KasI and XmaI sites.
  • a polylinker segment (SfPBXPS) was prepared by annealing complementary oligonucleotides (SfPBXPSsense and SfPBXPScomp). After digestion with KasI and XmaI, this segment was ligated into corresponding sites of pLOI4145 to produce pLOI4153.
  • the modified cat-sacB cassette in pLOI4152 was amplified by PCR using the JMcatsacBup3/down3 primer set.
  • this cassette was ligated into corresponding sites of pLOI4153 to produce pLOI4146.
  • pLOI4146 was digested with Pact and self-ligated to produce pLOI4154 (not shown), removing the cat-sacB cassette.
  • Two additional bases were inserted between the SfoI and Pad sites of pLOI4154 using mutagenic primers (JM4161 sense/comp) and linear plasmid amplification to produce pLOI4161.
  • a modified method was developed to delete E. coli chromosomal genes using a two-step homologous recombination process (Thomason et al., 2005). With this method, no antibiotic genes or scar sequences remain on the chromosome after gene deletion.
  • part of the target gene was replaced by a DNA cassette containing a chloramphenicol resistance gene (cat) and a levansucrase gene (sacB).
  • cat chloramphenicol resistance gene
  • sacB levansucrase gene
  • the cat-sacB cassette was replaced with native sequences omitting the region of deletion. Cells containing the sacB gene accumulate levan during incubation with sucrose and are killed. Surviving recombinants are highly enriched for loss of the cat-sacB cassette.
  • a cassette was constructed to facilitate gene deletions.
  • the cat-sacB region was amplified from pEL04 (Lee et al., 2001; Thomason et al., 2005) by PCR using the JMcatsacB primer set (Table 3), digested with NheI, and ligated into the corresponding site of pLOI3421 to produce pLOI4151.
  • the cat-sacB cassette was amplified by PCR using pLOI4151 (template) and the cat-up2/sacB-down2 primer set (EcoRV site included in each primer), digested with EcoRV, and used in subsequent ligations.
  • the mgsA gene and neighboring 500 bp regions were amplified using primer set mgsA-up/down and cloned into the pCR2.1-TOPO vector (Invitrogen) to produce plasmid pLOI4228.
  • a 1000-fold diluted preparation of this plasmid DNA served as a template for inside-out amplification using the mgsA-1/2 primer set (both primers within the mgsA gene and facing outward).
  • the resulting 4958 bp fragment containing the replicon was ligated to the amplified, EcoRV-digested cat-sacB cassette from pLOI4151 to produce pLOI4229.
  • pLOI4230 This 4958 bp fragment was also used to construct a second plasmid, pLOI4230 (phosphorylation and self-ligation).
  • pLOI4230 the central region of mgsA is absent (yccT′-mgsA′-mgsA′′-hel).
  • step I yccT′-mgsA′-cat-sacB-mgsA′′-helD′
  • step II yccT′-mgsA′-mgsA′′-helD′
  • recombinants were selected for chloramphenicol (40 mg l ⁇ 1 ) and ampicillin (20 mg l ⁇ 1 ) resistance on plates (30° C., 18 h). Three clones were chosen, grown in Luria broth with ampicillin and 5% w/v arabinose, and prepared for electroporation. After electroporation with the step II fragment, cells were incubated at 37° C. for 4 h and transferred into a 250-ml flask containing 100 ml of modified LB (100 mM MOPS buffer added and NaCl omitted) containing 10% sucrose.
  • modified LB 100 mM MOPS buffer added and NaCl omitted
  • clones were selected on modified LB plates (no NaCl; 100 mM MOPS added) containing 6% sucrose (39° C., 16 h). Resulting clones were tested for loss of ampicillin and chloramphenicol resistance. Construction was further confirmed by PCR analysis. A clone lacking the mgsA gene was selected and designated KJ070.
  • the poxB gene was deleted from KJ071 in a manner analogous to that used to delete the mgsA gene. Additional primer sets (poxB-up/down and poxB-1/2) used to construct the poxB deletion are included in Table 3 together with the corresponding plasmids (pLOI4274, pLOI4275, and pLOI4276). The resulting strain was designated KJ072.
  • the tdcDE gene and neighboring 1000 bp regions were amplified using tdcDEup/down primers and cloned into the pCR2.1-TOPO vector to produce plasmid pLOI4515.
  • a 1000-fold diluted preparation of this plasmid DNA served as a template for inside-out amplification using the tdcDEF7/R7 primers (both primers within the tdcDE gene and facing outward).
  • the resulting 6861 bp fragment containing the replicon was ligated to the amplified, SmaI/SfoI-digested cat-sacB cassette from pLOI4162 (JMcatsacBup3/down3 primers) to produce pLOI4516.
  • This 6861 bp fragment was also used to construct a second plasmid, pLOI4517 (kinase treated, self-ligation) containing a deletion of tcdD and tdcE.
  • the PCR fragments amplified from pLOI4516 and pLOI4517 (tdcDEup/down primers) were used to replace tdcDE region in KJ091.
  • the resulting clones were tested for loss of ampicillin and chloramphenicol resistance.
  • the aspC gene was deleted from KJ104 in a manner analogous to that used to delete the tdcDE gene. Additional primer sets (aspCup/down and aspC1/2) used to construct the aspC deletion are included in Table 7 together with the corresponding plasmids (pLOI4280, pLOI4281, and pLOI4282). The resulting strain was designated KJ110. Neither KJ098, nor KJ110 contain any intervening sequence within the respective deleted regions (tdcDE and aspC).
  • the E. coli strains XZ464, XZ465, and XZ466 were derived from the E. coli strain XZ632 using the genetic methods described above.
  • the relevant characteristic of the E. coli strains XZ464, XZ465, XZ466 and XZ632 are provided in the Table 15.
  • RNA purification was performed with RNeasy Mini columns (Qiagen), followed by digestion with DNaseI (Invitrogen). Reverse transcription with Superscript II (Invitrogen, Carlsbad Calif.) used 50 ng total RNA as template.
  • the production of the organic acid by the genetically engineered microorganism can be confirmed and quantified by using appropriate techniques well known in the art.
  • HPLC techniques can be used to measure the quantity of the organic acid produced by the selected clone.
  • the HPLC technology is also helpful in determining the purity of the organic acid produced by the selected clones.
  • Organic acids and sugars were determined by using high performance liquid chromatography (Grabar et al., 2006; Zhang et al., 2007).
  • Succinic acid and other organic acids present in the fermentation broth were analyzed on Agilent 1200 HPLC apparatus with BioRad Aminex HPX-87H column. BioRad Microguard Cation H + was used as a guard column.
  • the standards for HPLC analysis were prepared in 0.008N sulfuric acid.
  • the HPLC column temperature was maintained at 50° C.
  • Sulfuric acid at 0.008N concentration was used as a mobile phase at the flow rate of 0.6 ml/min. Quantification of various components was done by measuring their absorption at 210 nm.
  • Cells were harvested by centrifugation (8,000 ⁇ g for 5 min at 4° C.) during mid-log growth, washed with cold 100 mM Tris-HCl (pH 7.0) buffer, and resuspended in the same buffer (1 ml). After cellular disruption using a Fast Prep-24 (MP Biomedicals, Solon, Ohio), preparations were clarified by centrifugation (13,000 ⁇ g for 15 min). Protein was measured by the BCA method (Pierce, Rockford, Ill.) using bovine serum albumin as a standard.
  • PEP carboxylase activity was measured as described by Canovas and Kornberg (1969).
  • the reaction mixture contained 100 mM Tris-HCl buffer (pH8.0), 10 mM MgCl 2 , 1 mM DTT, 25 mM NaHCO 3 , 0.2 mM NADH, 20 U malate dehydrogenase, and crude extract.
  • the assay mixture was incubated for 15 min at 37° C. to activate the enzyme, after which the reaction was started by addition of 10 mM PEP.
  • PEP carboxykinase activity was measured as described by Van der Werf et al, (1997).
  • the reaction mixture contained 100 mM MES buffer (pH6.6), 10 mM MgCl 2 , 75 mM NaHCO 3 , 5 mM MnCl 2 , 50 mM ADP, 1 mM DTT, 0.2 mM NADH, 20 U malate dehydrogenase, and crude extract.
  • the reaction was started by addition of 10 mM PEP.
  • Malic enzyme activity was measured as described by Stols and Donnelly (1997).
  • the reaction mixture contained 100 mM Tris-HCl buffer (pH7.5), 25 mM NaHCO 3 , 1 mM MnCl 2 , 1 mM DTT, 0.2 mM NADH, and crude extract.
  • the reaction was started by addition of 25 mM pyruvate. This assay method was unsuitable for measurement of SfcA activity in wild type E. coli due to presence of lactate dehydrogenase.
  • ⁇ -galactosidase The activity of ⁇ -galactosidase was measured as described by Miller (1992). In all assays, one unit of activity represents the amount of enzyme required to oxidize or reduce 1 nmol of substrate per minute.
  • the KJ012 ( ⁇ ldhA::FRT ⁇ adhE::FRT ⁇ ackA::FRT) strain resulting from the insertional inactivation of ldhA, adhE and ack genes grew very poorly under anaerobic conditions in mineral salts medium containing 5% glucose (278 mM) and produced acetate instead of succinate as the primary fermentation product. Counter to expectations from rational design, succinate remained as a minor product. Molar yields of succinate based on metabolized glucose were unchanged as a result of these mutations. The succinate yield was found to be 0.2 mol succinate per mol glucose both for the parent and KJ012 strains during fermentation in NBS mineral salts medium containing 5% glucose.
  • NBS mineral salts medium contains all mineral nutrients needed for the growth of KJ012 by incubating under aerobic conditions (aerobic shaken flask; 5% glucose). In aerobic shaken flasks, cell yields for KJ012 were 5-fold higher than during anaerobic growth and 75% that of the E. coli C (parent) during anaerobic growth. These results also confirmed that all central biosynthetic pathways remain functional in KJ012.
  • levels of these gluconeogenic enzymes vary inversely with the availability of glucose and other metabolites (Goldie and Sanwal, 1980a; Wright and Sanwal, 1969; Sanwal and Smando, 1969a) and function in the reverse direction, decarboxylation (Keseler et al., 2005; Oh et al., 2002; Kao et al., 2005; Stols and Donnelly, 1997; Samuelov et al., 1991; Sanwal, 1970a; Delbaere et al., 2004; Goldie and Sanwal, 1980b; Sanwal and Smando, 1969b; Sanwal 1970b).
  • KJ012 Metabolic evolution of KJ012 was carried out by sequentially subculturing under various regimens using small, pH-controlled fermentors to improve the growth.
  • KJ012 was serially transferred in NBS glucose medium under fermentative conditions as rapidly as growth permitted ( FIG. 3A ; FIG. 4A ; FIG. 5 ). Growth remained slow with 4-5 days of required incubation between the first 9 transfers, then dramatically increased allowing transfers to be made at 24 h-intervals. This event was accompanied by an increase in acetate ( FIG. 4A ) with little improvement in succinate production.
  • KOH was replaced with a 1:1 mixture of 3M K 2 CO 3 and 6N KOH to provide additional carbon dioxide (100 mM initially added to all NBS mineral salts medium).
  • pflB pyruvate formatelyase
  • the upstream formate transporter (focA) in this operon was also deleted.
  • this deleted strain KJ032 did not grow without acetate confirming that this is the primary route for acetyl ⁇ CoA production in KJ017 ( FIG. 3C ).
  • Deletion of pflB is well-known to cause acetate auxotrophy under anaerobic conditions (Sawers and Bock, 1988). Growth and succinate production by KJ032 were restored by the addition of 20 mM acetate ( FIG.
  • lactate production by this pathway is indicative of methylglyoxal accumulation, an inhibitor of both growth and glycolysis (Egyud and Szent-Gyorgyi, 1966; Grabar et al., 2006; Hopper and Cooper, 1971).
  • KJ070 Production of methylglyoxal and lactate were eliminated by deleting the mgsA gene (methylglyoxal synthase) in KJ060 to produce KJ070 ( ⁇ ldhA::FRT ⁇ adhE::FRT ⁇ ackA::FRT ⁇ focA-pflB::FRT ⁇ mgsA).
  • Strain KJ070 was initially subcultured in 5% (w/v) glucose ( FIG. 3E , FIG. 4E , and FIG. 5 ). Deletion of mgsA is presumed to have increased glycolytic flux as evidenced by the accumulation of pyruvate in the medium (Table 4).
  • This increase in glycolytic flux may also be responsible for the further decline in the succinate/malate ratio due to increased production of oxaloacetate, an allosteric inhibitor of fumarate reductase (Iverson et al., 2002; Sanwal, 1970c).
  • Pyruvate oxidase represents a potential source of acetate and CO 2 during incubation under microaerophilic conditions (Causey et al., 2004). Although it should not function to oxidize pyruvate under anaerobic condition, poxB was targeted for gene deletion ( FIG. 6 ).
  • Strain KJ072 was subjected to 40 further rounds of metabolic evolution in AM1 medium, a lower salt medium, with 10% (w/v) glucose (Table 4; FIG. 3F , FIG. 4F , and FIG. 5 ). Improvements in growth, cell yield and succinate production were observed during these transfers. Malate, pyruvate and acetate levels also increased. A clone was isolated from the final transfer and designated KJ073 ( ⁇ ldhA::FRT ⁇ adhE::FRT ⁇ ackA::FRT ⁇ pflB::FRT ⁇ mgsA ⁇ poxB).
  • the KJ073 strain retained the phosphoenolpyruvate carboxykinase route for carboxylation (Table 5). In vitro activity of this strain was 45-fold higher than that of KJ012 and 10-fold higher than KJ017 providing further evidence for the tight coupling of energy conservation to succinate production and growth and further establishing the basis used for selection.
  • the pck and surrounding regions were cloned from KJ012 and KJ073, and sequenced. No changes were found in the coding region. Absent post-translational modifications, the catalytic properties of the enzyme should be unchanged.
  • a single mutation was detected in the pck promoter region, G to A at ⁇ 64 bp site relative to the translation start site. This mutation was behind the transcription start site which is ⁇ 139 bp site relative to the translational start site.
  • Rumen bacteria such as Actinobacillus succinogenes produce succinate as a primary product during glucose fermentation using the energy conserving phosphoenolpyruvate carboxykinase for carboxylation (Kim et al., 2004; McKinlay et al., 2005; McKinlay and Vieille, 2008). Reported activities for this organism are 5-fold those of KJ017 and half of that obtained by continued growth-based selection (metabolic evolution) of KJ073.
  • the studies reported herein demonstrate the development of succinate-producing strains of E. coli that resemble a rumen organism such as A.
  • FIG. 7 shows batch fermentations with KJ060 and KJ073, the two best biocatalysts for succinate production. Although growth was completed within the initial 48 h of incubation, succinate production continued for 96 h. One-third of succinate production occurred in the absence of cell growth. These strains produced succinate titers of 668-733 mM, with a molar yield of 1.2-1.6 based on glucose metabolized. With AM1 medium, yields were typically higher than with NBS mineral salts medium. Acetate, malate, and pyruvate accumulated as undesirable co-products and detracted from the potential yield of succinate (Table 4). The maximum theoretical yield of succinate from glucose and CO 2 (excess) is 1.71 mol per mole glucose based on the following equation:
  • E. coli has the native ability to metabolize all hexose and pentose sugars that are constituents of plant cell walls (Asghari et al., 1996; Underwood et al., 2004). Some strains of E. coli can also metabolize sucrose (Moniruzzaman et al., 1997). Strain KJ073 was tested for utilization of 2% sugars of hexoses and pentoses in serum tubes. In all cases, these sugars were converted primarily to succinate. Strain KJ073 also metabolized glycerol to succinate.
  • the fermentative metabolism of E. coli has been shown to be remarkably adaptable. Derivatives were engineered and evolved to circumvent numerous deletions of genes concerned with native fermentation pathways and increase fluxes through remaining enzymes to maintain redox balance, increase the efficiency of ATP production, and increase growth. Though much more challenging, cells can make such adaptive changes in mineral salts media while balancing carbon partitioning to provide all biosynthetic needs. After eliminating the primary routes for NADH oxidation (lactate dehydrogenase, alcohol dehydrogenase) and acetate production (acetate kinase), growth and ATP production remain linked to NADH oxidation and the production of malate or succinate for redox balance ( FIG. 1B ).
  • Anaerobic growth-based selections ensure redox balance and select for increased efficiency and increased rates of ATP production, the basis for increased growth. This selection for redox balance and ATP production cannot readily distinguish between malate and succinate as end products, since the precursors of both serve as electron acceptors.
  • the resulting strain, KJ073, produced 1.2 moles of succinate per mole of metabolized glucose (Jantama et al., 2008a) and uses a succinate pathway quite analogous to the rumen bacteria, Actinobacillus succinogenes (van der Werf et al., 1997) and Mannheimia succiniciproducens (Song et al., 2007).
  • Actinobacillus succinogenes van der Werf et al., 1997) and Mannheimia succiniciproducens (Song et al., 2007).
  • methods used to construct these gene deletions left a single 82 to 85 nucleotide-long genetic scar or FRT site in the region of each deleted gene (ackA, ldhA, adhE, ackA, focA-pflB).
  • the KJ091 strain is devoid of all foreign and synthetic DNA except for an 18-bp translational stop sequence within ackA. Succinate production by strain KJ091 was equivalent to that of KJ073 strain (Table 8). This strain was used as the parent for further improvements in succinate production.
  • a related enzyme with acetate kinase (and proprionate kinase) activity is encoded by tdcD but is typically produced only for the degradation of threonine (Hesslinger et al., 1998; Reed et al., 2003). It is possible that mutations occurring during selection have increased expression of tdcD as illustrated in FIG. 10 . During anaerobic growth with 10% (w/v) glucose, expression of tdcD could functionally replace ackA, increasing the production of acetate from acetyl ⁇ P.
  • the adjacent tdcE gene in the same operon is similar to pflB and encodes a pyruvate (and a-ketobutyrate) formatelyase activity that is co-expressed during threonine degradation (Hesslinger et al., 1998). It is possible that increased expression of this gene during anaerobic growth with 10% (w/v) glucose could increase the production of acetyl ⁇ CoA, the immediate precursor of acetyl ⁇ P, and waste reductant as formate ( FIG. 10 ). Both tdcD and tdcE (adjacent) were simultaneously deleted from KJ091 to produce KJ098.
  • KJ098 represents a significant improvement over KJ091, further reduction in acetate levels and further increases in succinate yields may be possible.
  • oxaloacetate is partitioned into the reduced product (malate) and oxidized intermediate (citrate) ( FIG. 9 ).
  • Citrate can be converted back to oxaloacetate and acetate by citrate lyase (citDEF) to recycle the intracellular OAA pool ( FIG. 10 ) for other metabolic functions (Nilekani et al., 1983).
  • citrate lyase is associated with growth on citrate (Lutgens and Gottschalk, 1980; Kulla and Gottschalk, 1977).
  • Citrate lyase is a multi-enzyme complex made up of three different polypeptide chains.
  • the a or large subunit is a citrate-ACP transferase that catalyzes the first step.
  • the ⁇ or medium subunit is a citryl-ACP lyase that catalyzes the second step.
  • the y or small subunit acts as an acyl-carrier protein and also carries the prosthetic group components. All three subunits are required for the reaction to take place (Quentmeier et al., 1987). The deletion of genes encoding one or more of these subunits would eliminate citrate lyase activity and may further reduce the level of acetate during succinate production.
  • citF gene was deleted from KJ098 to produce KJ104. This deletion, however, had no effect on acetate production or other succinate yield (Table 8). Since deletion of citF did not cause any reduction in acetate, this intermediate is presumed to arise from other pathways. For unknown reasons, deletion of citF adversely affected the growth of KJ104 (reduced cell yield by 22%) and increased the level of pyruvate at the end of fermentation by almost 50% in comparison to KJ098. However, the succinate yield, titer, average productivity, and acetate levels with KJ104 were comparable to those with KJ098 (Table 8).
  • Aspartate aminotransferase is a multifunctional enzyme that catalyzes the synthesis of aspartate, phenylalanine and other compounds by transamination.
  • L-aspartate is synthesized from oxaloacetate, an intermediate from PEP carboxylation, by a transamination reaction with L-glutamate.
  • aspartate participates in several other biosynthetic pathways. About 27 percent of the cellular nitrogen has been estimated to flow through aspartate (Reitzer, 2004).
  • Aspartate biosynthesis and succinate production share a common intracellular pool of oxaloacetate. Deletion of aspC could lead to increased succinate production but may also create an auxotrophic requirement that prevents anaerobic growth in minimal salts medium such as AM1.
  • This aspartate aminotransferase gene (aspC) was deleted from KJ104 to produce KJ110. Unexpectedly, the deletion of aspC had no effect on succinate yield or cell yield in KJ110 as compared to KJ104 (Table 8). Thus aspartase does not appear to divert significant levels of oxaloacetate away from succinate production in our strain. Alternative enzymes appear to be available that replace the biosynthetic needs formerly catalyzed by aspartate aminotransferase.
  • KJ122 produced excellent succinate yields (1.5 mol mol ⁇ 1 glucose) plus smaller amounts of acetate and pyruvate.
  • the maximum theoretical yield for succinate is 1.71 mol mol ⁇ 1 glucose and these 3-carbon intermediates represent an opportunity to further increase yield.
  • Pyruvate is presumed to accumulate from glycolysis as a metabolic overflow and may be related to acetate accumulation.
  • Acetyl ⁇ CoA is an allosteric regulator of many enzymes. The source of acetate and acetate kinase activity is unknown since genes encoding the two primary activities for acetate kinase (tdcD and ackA) have been deleted ( FIG. 9 and FIG. 10 ).
  • E. coli has the metabolic potential for four native carboxylation pathways that could be used to produce succinate ( FIGS. 2A-2D ).
  • the carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate (OAA) by phosphoenolpyruvate carboxylase (ppc) is recognized as the primary pathway for the fermentative production of succinate in E. coli ( FIG. 2A ).
  • PEP phosphoenolpyruvate
  • ppc phosphoenolpyruvate carboxylase
  • the three other carboxylation reactions are normally repressed by high concentration of glucose in the medium and also reported to function in the reverse direction for gluconeogenesis. (Samuelov et al., 1991; Oh et al., 2002; Stols & Donnelly, 1997).
  • the second and third carboxylation pathways ( FIGS. 2B and 2C ) use NADH-linked and NAPDH-linked malic enzymes (sfcA and maeB), respectively, to catalyze the reversible carboxylation of pyruvate to malate. Both pathways allow energy to be conserved as ATP during the pyruvate formation from PEP.
  • the fourth pathway uses PEP carboxykinase (pck) for the reversible carboxylation of PEP to OAA with the conservation of energy as ATP ( FIG. 2D ).
  • PEP carboxykinase PCK
  • PCK PEP carboxykinase
  • an analogous PEP carboxykinase is present at very high levels in succinate-producing rumen bacteria where it serves as the primary PEP carboxylation activity.
  • E. coli strain KJ073 was metabolically engineered by both targeted gene deletion and evolution for high growth rate and succinate production. (Jantama et al., 2008a). Genes encoding each of the four carboxylating enzymes (ppc, sfcA, maeB and pck) were individually deleted to identify the primary pathway for succinate production in the engineered strain KJ073 (Table 10). Deletion of the ppc gene encoding the primary carboxylating step in native E. coli (XZ320) did not alter succinate production.
  • This pck* mutation was present only in KJ060 and KJ073 strains showing high PEP carboxykinase activity and absent in KJ071. Loss of this mutation in KJ071 was accompanied by a 10-fold decrease in PEP carboxykinase activity to a level equivalent to that of 10017.
  • csrA The csrA system has also been reported to regulate the level of pck and other genes involved in glucose metabolism by altering mRNA stability. (Pernestig et al., 2003). However, no mutation was found in the sequences of genes involved in this regulatory system (csrA, csrB, csrC, csrD, uvrY or barA).
  • E. coli PEP carboxykinase activity is subject to glucose catabolite repression. (Goldie 1984). Two Crp-binding sites have been identified in the promoter (Ramseier et al 1995), quite distant from the point mutation in KJ060 and KJ073. Genes associated with catabolite repression (cyaA, crp) and glucose uptake by the phosphotransferase system (ptsH, ptsI, err, ptsG) were sequenced (upstream region through terminator). Only one mutation was found, a frame-shift mutation in the carboxy-terminal region of ptsI (single base deletion at position 1,673) in strains KJ060, KJ071, and KJ073.
  • the gene ptsI encodes PEP-protein phosphotransferase, a general (non sugar-specific) component of the phosphoenolpyruvate-dependent sugar phosphotransferase system.
  • This major carbohydrate active-transport system catalyzes the phosphorylation of incoming sugar substrates concomitantly with their translocation across the cell membrane.
  • PEP-protein phosphotransferase transfers the phosphoryl group from phosphoenolpyruvate (PEP) to the phosphoryl carrier protein (HPr). (See, e.g., UniProtKB, Accession Number P08839).
  • a frame-shift mutation in ptsI (single base-pair deletion of position 1673 bp) was found to have occurred during the metabolic evolution of KJ060 from KJ017.
  • the carboxy-terminal 175 bp of ptsI was deleted in KJ017 and KJ060 to produce XZ613 and XZ615, respectively.
  • the ptsI deletion in KJ060 (XZ615) had no effect on growth and PEP carboxykinase activities as expected.
  • the ptsI mutation found in KJ060, KJ071, and KJ073 would be expected to inactivate the PEP-dependent phosphotransferase system for glucose.
  • Alternative glucose uptake systems such as GalP have been shown to restore glucose uptake in pts mutants. (Wang et al., 2006; Yi et al., 2003). Expression of galP was increased by 5-fold to 20-fold in these improved strains ( FIG. 12B ) as compared to the KJ012 and ATCC 8739, with a smaller increase in glucokinase (glk).
  • Carbon fluxes at the PEP node serve to limit the amount of succinate produced for redox balance during the anaerobic fermentation of glucose ( FIG. 13 ). In addition, these fluxes must provide sufficient energy (ATP) for growth, maintenance, and precursors for biosynthesis. Rumen bacteria that produce succinate as the dominant product use an energy-conserving PEP carboxykinase for OAA production ( FIG. 2D ). (Van der Werf et al., 1997; Kim et al., 2004). Native strains of E. coli produce succinate as a minor product and use PEP carboxylase (ppc) ( FIG. 2A ).
  • PEP carboxylase is essentially irreversible due to the energy loss associated with release of inorganic phosphate.
  • Previous studies have shown that overexpression of the native ppc gene in E. coli resulted in higher specific succinate production (Millard et al., 1996) and higher specific growth rate due to increased carboxylation of PEP to oxaloacetate. (Farmer & Liao, 1997).
  • PEP is required for the native glucose transport system, over expressing ppc also decreased the rate of glucose uptake without significantly increasing succinate yield. (Chao & Liao, 1993; Gokarn et al., 2000).
  • FIGS. 2B-2D Native E. coli strains have three alternative carboxylation pathways ( FIGS. 2B-2D ) that typically function only in the reverse direction for gluconeogenesis. (Samuelov et al., 1991; Kao et al., 2005; Oh et al., 2002; Stols & Donnelly, 1997). All three would allow the conservation of energy from PEP as ATP. With succinate as the sole route for NADH oxidation, growth-based selection (metabolic evolution) resulted in strains KJ017, KJ060, KJ071, and KJ073 with improvements in growth (cell yield) and succinate production.
  • Increased expression of pck in E. coli resulted in increased carbon flow into the 4-carbon intermediate OAA for succinate production (redox balance), increased succinate production, and increased the net production of ATP for growth and maintenance.
  • At least three events contributed to increased transcription of pck 1) loss of Crp-mediated glucose-repression; 2) gain of glucose-activation; and 3) a single base change in the upstream region of pck.
  • Each of these events provided a basis for selection through metabolic evolution by increasing the level of PEP carboxykinase, increasing the flow of carbon into succinate, and increasing the conservation of metabolic energy as ATP.
  • strains KJ060, KJ071, and KJ073 which may potentially assist in the recruitment of PEP carboxykinase as the primary route for fermentative succinate production.
  • the PEP-dependent phosphotransferase system is the primary glucose uptake system in native strains of E. coli . During transport, half of the PEP produced from glucose is used for uptake and phosphorylation, limiting metabolic options for redox balance and ATP production.
  • the improved strains contained a frame-shift mutation in ptsI that inactivated this uptake system. Expression of galP encoding a proton symporter which can transport glucose was increased by up to 20-fold.
  • PEP is conserved to eliminate the need for energy-expensive regeneration (2-ATP equivalents) by using glucose permeases (and glucokinase) rather than the PEP-dependent phosphotransferase system for glucose uptake.
  • glucose permeases and glucokinase
  • the phosphotransferase system is widely used for glucose uptake.
  • the carboxylation product, OAA is an important intermediate in cell metabolism that serves as a precursor for many other important fermentation products such as malic acid, fumaric acid, aspartic acid, lysine, threonine, methionine, among others.
  • energy conservation strategies illustrated herein may be used to develop and improve biocatalysts for the production of many industrially important chemicals.
  • the pck gene (ribosomal binding site, coding and terminator region) was amplified and cloned into pCR2.1-TOPO to produce pLOI4677 with pck expression under control of the lac promoter.
  • This plasmid was transformed into ATCC 8739.
  • the native pck gene in ATCC 8739 was replaced with the mutant pck* gene to construct XZ632. Glucose fermentation was examined in both strains using NBS mineral salts medium.
  • the pck* mutation was tested in combination with other mutations that eliminated pathways for NADH oxidation (Table 18). The combined action of mutations to eliminate competing routes for NADH oxidation and increased PCK activity were also insufficient to substantially redirect glucose carbon to succinate.
  • the phosphoenolpyruvate-dependent phosphotransferase system is the primary mechanism for glucose uptake in E. coli and an integral part of the glucose catabolite repression system. (Keseler et al., 2005; Postma et al., 1996). As described in the Example 3 above, a frame-shift mutation within the carboxyl end of ptsI (single base-pair deletion at 1673 bp position) was discovered in the succinate producing strains KJ060, KJ071 and KJ073 as a result of metabolic evolution. This mutation would be expected to disrupt function of the phosphotransferase system. (Postma et al., 1996).
  • the PEP-dependent phosphotransferase system shuttles phosphate from PEP to PtsI, then PtsH, then PtsG, and then to glucose to form glucose 6-phosphate.
  • Table 19 shows that in a strain containing the pck* mutation that increases the level of phosphoenolpyruvate carboxykinase, a second mutation in any one of the Pts genes involved in this phosphate relay system (ptsI, ptsH or ptsG) results in a similar dramatic shift in carbon flow into succinate as the dominant fermentation product.
  • deletion of the whole ptsI gene or a truncation of the carboxyterminus of the ptsI gene was superior to the ptsG and ptsH deletions.
  • the truncation of the ptsI carboxyterminus produced the highest yield and titer of succinate, slightly better than the complete deletion of ptsI.
  • Other mutations such as insertions, deletions, and frame-shifts that lead to inactive gene products would be expected to have similar effects.
  • strain XZ647 (pck* ⁇ ptsI) produced succinate as the dominant fermentation product, significant levels of unwanted co-products (lactate, ethanol, formate, and acetate) were also produced (Table 18).
  • the resulting strain, (XZ721) produced high levels of succinate with a molar yield of over 1.2 mol succinate per mol glucose.
  • Succinate typically represents a minor product of glucose fermentation in E. coli .
  • Most of the glucose carbon is converted to ethanol and lactate with smaller amounts of formate and acetate using alternative NADH-oxidizing pathways ( FIG. 14 ).
  • Derivatives of E. coli have been constructed to improve succinate production for more than a decade with variable success.
  • Donnelly et al. U.S. Pat. No. 5,770,435; Gokarn et al., 2000; Gokarn et al., U.S. Pat. No. 6,455,284; Millard et al., 1996; San et al., U.S. Pat. No.
  • Target genes for deletion were selected primarily based on inspection of the pathway ( FIG. 14 ). However, successes with this strategy have been limited to complex medium and two-step (aerobic growth phase followed by anaerobic production phase) processes.
  • Donnelly et al. U.S. Pat. No. 5,770,435; Gokarn et al., U.S. Pat. No. 6,455,284; Millard et al., 1996; San et al., U.S. Pat. No. 7,223,567; Sanchez et al., 2005b; Sanchez et al., 2005a; Vemuri et al., 2002a; Wu et al., 2007).
  • this invention provides a new strategy to construct strains for succinate production in mineral salts medium. No mutations were required in genes encoding the E. coli mixed acid fermentation pathway ( FIG. 14 ) to substantially redirect glucose carbon to succinate. In this invention, the combination of two core changes in peripheral pathways resulted in a five-fold increase in succinate yield.
  • the two core changes that are required for succinate production area 1) increased expression of the energy conserving (gluconeogenic) phosphoenolpyruvate carboxykinase to replace the native fermentative phosphoenolpyruvate carboxylase (energy wasting) and 2) replacement of the glucose phosphoenolpyruvate-dependent phosphotransferase system with an alternative permease such as galP and ATP-dependent phosphorylation (glk).
  • these changes increased net ATP production for growth, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production.
  • acetyl-CoA is an essential metabolite for biosynthesis that is produced primarily by pflB during fermentative growth. This function is presumed to be replaced in pflB mutants by native expression of the pyruvate dehydrogenase complex (aceEF, lpd), an enzyme that typically serves as the dominant route for acetyl-CoA production during oxidative metabolism. (Kim et al., 2007).
  • the resulting succinate pathway in E. coli strains optimally engineered for succinate production in mineral salts medium FIG.
  • gldA and the PEP-dependent phosphotransferase pathway previously regarded as cryptic comprise an important functional route for glycerol catabolism ( FIG. 15 ).
  • glycerol enters the cells by facilitated diffusion.
  • part of the glycerol is immediately phosphorylated followed by oxidation to DHAP, the pathway that is widely regarded as the standard pathway for glycerol metabolism in E. coli .
  • DHAP serves as the common entry point to central metabolism for both uptake and activation systems.
  • Table 20 clearly demonstrates the effectiveness of combining the core mutations identified for succinate production with glucose for succinate production from glycerol in mineral salts medium (NBS) without the addition of complex nutrients.
  • deletion of pflB alone reduced succinate production below that of the wild type parent
  • Subsequent addition of a mutation in ptsI further increased the succinate yield to 0.8 mol of succinate/mol glycerol, 80% of the maximum theoretical yield.
  • FIG. 15 shows a combination of the generally accepted pathway for glycerol catabolism (Lin, 1996) and the mixed acid fermentation pathway (Bock and Sawers, 1996) in wild type E. coli .
  • this pathway all ATP produced would be consumed by glycerol phosphorylation if succinate is produced as a sole product. No ATP would be available for growth.
  • deletion of pflB would be expected to increase succinate production by increasing the availability of reductant and intermediates. In contrast to this expectation, a decrease in succinate production was observed with the deletion of pflB gene (Table 20).
  • This minor pathway first reduces intracellular glycerol to dihydroxyacetone (DHA) with GldA then uses PtsH (ptsH) and EI (ptsI) as phosphate carriers to couple phosphoenolpyruvate to the phosphorylation of intracellular DHA to produce dihydroxyacetone-phosphate (DHAP).
  • PtsH PtsH
  • ptsI EI
  • DHAP dihydroxyacetone-phosphate
  • glycerol dehydrogenase (gldA) and PTS phospho-relay system phosphoenolpyruvate, PtsH, PtsI
  • DHAP dihydroxyacetone phosphate
  • DHA dihydroxyacetone
  • lactis pyc MgCO 3 200 mg/l ampicillin, and 1 mM IPTG. Dual phase with 100% CO 2 headspace, 168 h incubation a Abbreviations: CSL, corn steep liquor; YE, yeast extract; NR, not reported. b Average volumetric productivity is shown in brackets [g l ⁇ 1 h ⁇ 1 ] beneath succinate titer. c The molar yield was calculated based on the production of succinate from metabolized sugar during both aerobic and anaerobic conditions. Biomass was generated predominantly during aerobic growth. Succinate was produced primarily during anaerobic incubation with CO 2 , H 2 , or a mixture of both.
  • b Cell yield estimated from optical density (3 OD 550 nm 1 g l ⁇ 1 CDW).
  • c Succinate yields were calculated based on glucose metabolized.
  • d Average volumetric productivity was calculated for total incubation time.
  • e Abbreviations: suc, succinate; mal, malate; pyr, pyravate; ace, acetate; lac, lacate; for, formate.
  • f Average of 3 or more fermentations with standard deviations.
  • g Dash indicates absence of product.
  • Aerobic shaken flask 100 rpm; 100 ml NBS, 250-ml flask.
  • coli C (using JMackA-F1/R1 primers) Disclosed cloned into pCR2.1-TOPO vector herein pLOI4159 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4158 (using herein JMackAup1/down1) pLOI4160 PacI digestion of pLOI4159, then self-ligated Disclosed herein pLOI4515 bla kan; tdcG′-tdcFED-tdcC′ (PCR) from E.
  • pLOI4159 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4158 (using herein JMackAup1/down1) pLOI4160 PacI digestion of pLOI
  • coli C (using tdcDE- Disclosed up/down primers) cloned into pCR2.1-TOPO vector herein pLOI4516 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4515 (using tdcDE- herein F7/R7 primers) pLOI4517 PCR fragment amplified inside-out product from pLOI415 (using Disclosed tdcDE-F7/R7 primers), kinase treated, then self-ligated herein pLOI4629 bla kan; citF (PCR) from E.
  • pLOI4516 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4515 (using tdcDE
  • coli C (using citF-up2/down2 primers) Disclosed cloned into pCR2.1-TOPO vector herein pLOI4630 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4629 (using citF-2/3 herein primers) pLOI4631 PacI digestion of pLOI4630, then self-ligated Disclosed herein pLOI4280 bla kan; aspC (PCR) from E.
  • coli C (using aspC-up/down primers) Disclosed cloned into pCR2.1-TOPO vector herein pLOI4281 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4280 (using aspC-1/2 herein primers) pLOI4282 PCR fragment amplified inside-out product from pLOI4280 (using Disclosed aspC-1/2 primers), kinase treated, then self-ligated herein pLOI4283 bla kan; sfcA (PCR) from E.
  • pLOI4281 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4280 (using aspC-1/2 herein primers)
  • coli C (using sfcA-up/down primers) Disclosed cloned into pCR2.1-TOPO vector herein pLOI4284 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4283 (using sfcA-1/2 herein primers) pLOI4285 PacI digestion of pLOI4284, then self-ligated Disclosed herein pLOI4710 bla kan; ackA-pta (PCR) from E.
  • pLOI4284 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4283 (using sfcA-1/2 herein primers)
  • pLOI4285 PacI digestion of pLOI4284, then self
  • coli C (using ackA-up/pta-down Disclosed primers) cloned into pCR2.1-TOPO vector herein pLOI4711 SmaI/SfoI digested cat-sacB cassette from pLOI4162 cloned into the Disclosed PCR amplified inside-out product from pLOI4710 (using ackA-2/pta- herein 2 primers)
  • pLOI4712 PacI digestion of pLOI4711, then self-ligated Disclosed herein pLOI4413 bla kan; ychE′-adhE-ychG′ (PCR) from E.
  • coli C (using up/down- Disclosed adhE primers) cloned into pCR2.1-TOPO vector herein pLOI4419 PCR fragment amplified inside-out product from pLOI4413 (IO- Disclosed adhE-up/down using primers), kinase treated, then self-ligated herein pLOI4415 bla kan; ycaO′-focA-pflB-pflA′ (PCR) from E.
  • coli C (using up- Disclosed focA/Mid-pflA primers) cloned into pCR2.1-TOPO vector herein pLOI4421 PCR fragment amplified inside-out product from pLOI4415 (using Disclosed IO-ycaO-up/IO-midpflB-down primers), kinase treated, then self- herein ligated pLOI4430 bla kan; hslJ′-ldhA-ydbH′ (PCR) from E.
  • PEP-carboxykinase Strain Genotype (U/mg protein) KJ017 ⁇ pck + , ptsI + , cra + 700 XZ618 KJ017, pck* 6,419 XZ613 KJ017, )ptsI 614 XZ626 KJ017, )cra 508 KJ060 ⁇ ⁇ pck*, ptsI ⁇ , - cra + 8,363 XZ622 ⁇ KJ060, - pck + 1,103 XZ615 KJ060, )ptsI 10,309 XZ627 KJ060, )cra 7181 KJ071 ⁇ ⁇ ptsI ⁇ , - pck + , cra + 679 XZ620 KJ071, pck* 6,193 KJ073 ⁇
  • ptsI ⁇ refers to a frame-shift mutation, a single base deletion in the carboxy-terminal region.
  • Plasmids and primers used in Example 3 Plasmids pCR2.1-TOPO bla kan; TOPO TA cloning vector pLOI4162 bla cat; plasmid to provide cat-sacB cassette sfcA deletion pLOI4283 bla kan; sfcA (PCR) from ATCC 8739 cloned into pCR2.1-TOPO vector pLOI4284 cat-sacB cassette (SmaI-SfoI fragment of pLOI4162) cloned into sfcA of pLOI4284 pLOI4285 PacI digestion of pLOI4284, and self-ligated ppc deletion pLOI4264 bla kan; ppc (PCR) from ATCC 8739 cloned into pCR2.1-TOPO vector pLOI4265 cat-sacB cassette (SmaI-SfoI fragment of pLOI4162) clon
  • pck* denotes mutated form of pck (G to A at ⁇ 64 relative to the ATG start).
  • ⁇ ptsI denotes deletion of the carboxy-terminal 175 bp of ptsI gene
  • ⁇ ptsI-W denotes deletion of the whole ptsI gene.
  • Succinate yield was calculated as mole of succinate produced per mol glucose metabolized.
  • 3 Fermentations were carried out in NBS medium with 5% glucose and 100 mM potassium bicarbonate at 37° C., pH 7.0, 150 rpm.
  • Suc succinate; Ace, acetate; Mal, malate; Pyr, pyruvate; Lac, lactate; For, formate; EtOH, ethanol.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US13/256,460 2009-04-02 2010-04-02 Engineering the pathway for succinate production Abandoned US20120058530A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/256,460 US20120058530A1 (en) 2009-04-02 2010-04-02 Engineering the pathway for succinate production

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US16609309P 2009-04-02 2009-04-02
US13/256,460 US20120058530A1 (en) 2009-04-02 2010-04-02 Engineering the pathway for succinate production
PCT/US2010/029728 WO2010115067A2 (en) 2009-04-02 2010-04-02 Engineering the pathway for succinate production

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
PCT/US2008/057439 Continuation-In-Part WO2008115958A2 (en) 2007-03-20 2008-03-19 Materials and methods for efficient succinate and malate production
US12/529,826 Continuation-In-Part US8691539B2 (en) 2007-03-20 2008-03-19 Materials and methods for efficient succinate and malate production
PCT/US2010/029728 A-371-Of-International WO2010115067A2 (en) 2007-03-20 2010-04-02 Engineering the pathway for succinate production

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/705,011 Continuation US10287611B2 (en) 2007-03-20 2015-05-06 Engineering the pathway for succinate production

Publications (1)

Publication Number Publication Date
US20120058530A1 true US20120058530A1 (en) 2012-03-08

Family

ID=42828953

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/256,460 Abandoned US20120058530A1 (en) 2009-04-02 2010-04-02 Engineering the pathway for succinate production
US14/705,011 Expired - Fee Related US10287611B2 (en) 2007-03-20 2015-05-06 Engineering the pathway for succinate production

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/705,011 Expired - Fee Related US10287611B2 (en) 2007-03-20 2015-05-06 Engineering the pathway for succinate production

Country Status (11)

Country Link
US (2) US20120058530A1 (ko)
EP (1) EP2414505B1 (ko)
JP (2) JP2012522515A (ko)
KR (2) KR20120025450A (ko)
CN (1) CN102803470A (ko)
AU (2) AU2010232533A1 (ko)
BR (1) BRPI1009992A2 (ko)
CA (1) CA2756705C (ko)
MX (1) MX337341B (ko)
SG (1) SG175002A1 (ko)
WO (1) WO2010115067A2 (ko)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130157330A1 (en) * 2010-09-01 2013-06-20 University Of Florida Research Foundation, Inc. L-malate production by metabolically engineered escherichia coli
WO2014004753A1 (en) * 2012-06-29 2014-01-03 Archer Daniels Midland Company Betaine enhancement of fermentation to make c4 diacids
WO2014018902A2 (en) * 2012-07-26 2014-01-30 Joule Unlimited Technologies, Inc. Methods and compositions for the augmentation of pyruvate and acetyl-coa formation
WO2015013334A1 (en) * 2013-07-23 2015-01-29 Myriant Corporation Method of producing succinic acid and other chemicals using facilitated diffusion for sugar import
CN105062942A (zh) * 2015-08-26 2015-11-18 逢甲大学 可生产正丁醇的菌株及其应用
US20160097064A1 (en) * 2013-05-24 2016-04-07 Tianjin Institute Of Industrial Biotechnology, Chinese Academy Of Sciences Recombinant escherichia coli for producing succinic acid and application thereof
US20170101638A1 (en) * 2013-03-15 2017-04-13 Cargill, Incorporated Acetyl-coa carboxylases
US9970016B2 (en) 2015-11-12 2018-05-15 Industrial Technology Research Institute Genetic engineered bacteria and methods for promoting production of succinic acid or lactic acid
US10301653B2 (en) * 2015-07-06 2019-05-28 Wisconsin Alumni Research Foundation Microorganisms that co-consume glucose with non-glucose carbohydrates and methods of use
US10337038B2 (en) 2013-07-19 2019-07-02 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
US10465213B2 (en) 2012-08-10 2019-11-05 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
US10494654B2 (en) 2014-09-02 2019-12-03 Cargill, Incorporated Production of fatty acids esters
CN111411062A (zh) * 2020-05-07 2020-07-14 广东省农业科学院植物保护研究所 一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用
CN112300968A (zh) * 2020-11-19 2021-02-02 河南巨龙生物工程股份有限公司 产环磷腺苷的节杆菌及其应用
CN113278567A (zh) * 2020-02-20 2021-08-20 杭州立丞生物科技有限公司 可改善菌株以提升生产重组蛋白质的方法
US11345938B2 (en) 2017-02-02 2022-05-31 Cargill, Incorporated Genetically modified cells that produce C6-C10 fatty acid derivatives
US11408013B2 (en) 2013-07-19 2022-08-09 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products

Families Citing this family (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011063157A2 (en) 2009-11-18 2011-05-26 Myriant Technologies Llc Organic acid production in microorganisms by combined reductive and oxidative tricarboxylic acid cycle pathways
US10017793B2 (en) 2009-11-18 2018-07-10 Myriant Corporation Metabolic evolution of Escherichia coli strains that produce organic acids
CN102666838B (zh) * 2009-11-18 2018-04-10 麦兰特公司 用于高效产生化学品的工程化微生物
CA2775971C (en) 2009-11-18 2017-05-02 Myriant Technologies Llc Metabolic evolution of escherichia coli strains that produce organic acids
CN102781900A (zh) 2009-12-31 2012-11-14 麦兰特公司 从含有琥珀酸铵的发酵液纯化琥珀酸
WO2012082720A2 (en) 2010-12-13 2012-06-21 Myriant Corporation Method of producing succinic acid and other chemicals using sucrose-containing feedstock
WO2013009679A2 (en) 2011-07-08 2013-01-17 University Of Florida Research Foundation, Inc. Over-expression of a putative oxidoreductase (ucpa) for increasing furfural or 5-hydroxymethylfurfural tolerance
EP2734615B1 (en) 2011-07-22 2019-12-25 PTT Global Chemical Public Company Limited Fermentation of glycerol to organic acids
CN102286415B (zh) * 2011-09-07 2014-03-05 天津工业生物技术研究所 琥珀酸高产菌株及其应用
WO2013096602A1 (en) 2011-12-20 2013-06-27 Novomer, Inc. Methods for polymer synthesis
MY182946A (en) * 2012-09-14 2021-02-05 Ptt Global Chemical Public Co Ltd Production of organic acids by fermentation at low ph
CN103981203B (zh) 2013-02-07 2018-01-12 中国科学院天津工业生物技术研究所 5‑氨基乙酰丙酸高产菌株及其制备方法和应用
US20140296571A1 (en) * 2013-03-28 2014-10-02 The Procter & Gamble Company Microorganisms And Methods For Producing Propionic Acid
CN104178442B (zh) 2013-05-24 2017-10-31 中国科学院天津工业生物技术研究所 含有突变的lpdA基因的大肠杆菌及其应用
CN103243064B (zh) * 2013-05-28 2015-04-22 山东大学 一种大肠杆菌工程菌株及其好氧-微好氧-厌氧全阶段发酵生产琥珀酸的应用
CN103320367B (zh) * 2013-07-10 2014-12-31 南京工业大学 一株厌氧利用合成培养基高产丁二酸大肠杆菌的筛选及其应用
KR102113254B1 (ko) 2013-08-23 2020-05-21 삼성전자주식회사 1,4―ｂｄｏ 생산을 위한 유전자 스크리닝 방법
US10035749B2 (en) 2013-09-03 2018-07-31 Myriant Corporation Process for manufacturing acrylic acid, acrylonitrile and 1,4-butanediol from 1,3-propanediol
TW201538477A (zh) 2013-12-06 2015-10-16 Myriant Corp 製備丁二酸酯之方法
CN104894041B (zh) * 2014-03-03 2018-05-22 逢甲大学 可提升甘油代谢的菌株及其应用
KR102308323B1 (ko) * 2014-05-08 2021-10-06 바스프 에스이 수크로스 상의 정밀 화학물질의 개선된 생산을 위한 유전자 변형된 미생물
JP6319230B2 (ja) * 2015-08-24 2018-05-09 コニカミノルタ株式会社 有機エレクトロルミネッセンス素子用の芳香族複素環誘導体、それを用いた有機エレクトロルミネッセンス素子、照明装置及び表示装置
JP6319231B2 (ja) * 2015-08-24 2018-05-09 コニカミノルタ株式会社 有機エレクトロルミネッセンス素子用の芳香族複素環誘導体、それを用いた有機エレクトロルミネッセンス素子、照明装置及び表示装置
CN105779513B (zh) * 2016-05-10 2019-12-06 华东理工大学 利用重组大肠杆菌以甘油为碳源发酵生产琥珀酸的方法
CN110035991B (zh) * 2017-04-04 2022-06-17 Nnb营养品美国有限公司 一步发酵制备(r)-3-羟基丁酸或其盐
CN107012160A (zh) * 2017-05-15 2017-08-04 天津大学 高产琥珀酸的谷氨酸棒杆菌菌株及构建方法及应用
US11891378B2 (en) 2017-12-27 2024-02-06 St. Jude Children's Research Hospital, Inc. Small molecule modulators of pantothenate kinases
EP3731859A4 (en) 2017-12-27 2021-09-22 St. Jude Children's Research Hospital, Inc. METHODS OF TREATMENT FOR DISORDERS ASSOCIATED WITH CASTOR
KR20200115527A (ko) * 2018-01-23 2020-10-07 더 제너럴 하스피탈 코포레이션 미토콘드리아 기능 개선을 위한 조성물 및 방법
CN110527692B (zh) * 2018-05-25 2021-11-16 中国科学院微生物研究所 产l-鼠李糖的工程菌及其构建方法与应用
CN109929862B (zh) * 2019-03-14 2022-09-16 云南农业大学 一种从反刍动物瘤胃宏转录组数据筛选纤维素酶基因进行克隆的方法
CN110862975B (zh) * 2019-12-18 2021-08-31 西南大学 柑橘果胶乙酰酯酶CsPAE及其编码基因和应用
CN111826323A (zh) * 2020-08-05 2020-10-27 福建洛东生物技术有限公司 一种枯草芽孢杆菌及其制剂与应用
CN112063660B (zh) * 2020-09-29 2021-05-18 清远希普生物科技有限公司 一种提高枯草芽孢杆菌产酸量的工艺
CN112239738B (zh) * 2020-10-29 2022-11-25 江南大学 一株产琥珀酸的大肠杆菌及其应用
EP4262814A1 (en) * 2020-12-16 2023-10-25 St. Jude Children's Research Hospital, Inc. Methods of treating disorders associated with castor
CN112574925A (zh) * 2020-12-30 2021-03-30 广西科学院 一株耐受糠醛的产琥珀酸放线杆菌gxas-137fm及其选育方法与应用
FR3125046B1 (fr) 2021-07-09 2024-05-10 Snf Sa Procédé d’obtention de monomere N-vinylpyrrolidone biosourcé
CN113980868B (zh) * 2021-12-02 2023-02-03 广西科学院 一株耐受五羟甲基糠醛的产琥珀酸放线杆菌及其选育方法和应用

Family Cites Families (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162516A (en) 1988-05-31 1992-11-10 University Of Florida Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis
US5000000A (en) 1988-08-31 1991-03-19 University Of Florida Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes
US5028539A (en) 1988-08-31 1991-07-02 The University Of Florida Ethanol production using engineered mutant E. coli
WO1993018144A1 (en) 1992-03-05 1993-09-16 The Trustees Of Columbia University Of The City Of New York Recombination activating gene deficient animal
US5504004A (en) 1994-12-20 1996-04-02 Michigan Biotechnology Institute Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms
US6358714B1 (en) * 1995-04-03 2002-03-19 The Nutrasweet Company Materials and methods for the production of D-phenylalanine
US5869301A (en) 1995-11-02 1999-02-09 Lockhead Martin Energy Research Corporation Method for the production of dicarboxylic acids
US6159738A (en) 1998-04-28 2000-12-12 University Of Chicago Method for construction of bacterial strains with increased succinic acid production
US6607885B1 (en) 1999-10-15 2003-08-19 E. I. Du Pont De Nemours And Company Method for high-density microarray medicated gene expression profiling
US6911329B2 (en) 2000-09-23 2005-06-28 Degussa Ag Process for the fermentative preparation of D-pantothenic acid using coryneform bacteria
US6743610B2 (en) 2001-03-30 2004-06-01 The University Of Chicago Method to produce succinic acid from raw hydrolysates
US7145058B2 (en) 2002-03-27 2006-12-05 Council Of Scientific And Industrial Research Efficient method of preventing growth of microbial genetic transformant after transformation
US20040152159A1 (en) 2002-11-06 2004-08-05 Causey Thomas B. Materials and methods for the efficient production of acetate and other products
RU2268300C2 (ru) * 2003-04-07 2006-01-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИЙ, ОБЛАДАЮЩИХ ПОВЫШЕННОЙ ЭКСПРЕССИЕЙ ГЕНА pckA
US7244610B2 (en) 2003-11-14 2007-07-17 Rice University Aerobic succinate production in bacteria
FR2864967B1 (fr) 2004-01-12 2006-05-19 Metabolic Explorer Sa Microorganisme evolue pour la production de 1,2-propanediol
US7098009B2 (en) 2004-03-04 2006-08-29 University Of Florida Research Foundation, Inc. Production of chemicals from lignocellulose, biomass or sugars
PL1751292T3 (pl) * 2004-05-03 2011-01-31 Ut Battelle Llc Sposób wytwarzania kwasu bursztynowego z surowych hydrolizatów
JP5572279B2 (ja) 2004-05-20 2014-08-13 味の素株式会社 コハク酸生産菌及びコハク酸の製造方法
EP3130676A1 (en) * 2004-08-27 2017-02-15 Rice University Mutant e. coli strain with increased succinic acid production
CN101023178A (zh) * 2004-09-17 2007-08-22 莱斯大学 高琥珀酸产出细菌
PL1760156T3 (pl) 2005-08-10 2013-05-31 Univ Florida Materiały i sposoby wydajnej produkcji kwasu mlekowego
US20070111294A1 (en) * 2005-09-09 2007-05-17 Genomatica, Inc. Methods and organisms for the growth-coupled production of succinate
US20070072280A1 (en) 2005-09-19 2007-03-29 University Of Florida Research Foundation, Inc. Materials and methods for the efficient production of xylitol
WO2007115228A2 (en) * 2006-03-31 2007-10-11 Rice University Anaerobic fermentation of glycerol
CN101688196B (zh) * 2007-03-20 2015-01-07 佛罗里达大学研究基金公司 用于有效生产琥珀酸和苹果酸的材料和方法
WO2008119009A2 (en) 2007-03-27 2008-10-02 University Of Florida Research Foundation, Inc. Materials and methods for efficient alanine production
KR101103839B1 (ko) 2007-07-12 2012-01-06 한국과학기술원 순수 숙신산 생성 변이균주 및 이를 이용한 숙신산제조방법
WO2009072562A1 (ja) * 2007-12-06 2009-06-11 Ajinomoto Co., Inc. 有機酸の製造方法
BR112013012603A2 (pt) 2010-11-22 2016-08-09 Univ Florida Res Foudation Inc engenharia de bacillus coagulans termotolerante para a produção de ácido d(-)-láctico

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Gosset, G., Microbial Cell Factories 4:14, 2005 *
Hasona et al., Journal of Bacteriology 186(22):7593-7600, 2004 *
Kozak, M., Gene 234:187-208, 1999 *
Kwon et al., J. Microbiol. Biotechnol. 16(9):1448-1452, 2006 *
Onuffer et al., Protein Science 4:1750-1757, 1995 *
Sousa et al., Microbiology 148(Pt5):1291-1303, 2002 *
Zhou et al., Cell Mol Life Sci 63(19-20):2260-2290, 2006 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130157330A1 (en) * 2010-09-01 2013-06-20 University Of Florida Research Foundation, Inc. L-malate production by metabolically engineered escherichia coli
US9187772B2 (en) * 2010-09-01 2015-11-17 University Of Florida Research Foundation, Inc. L-malate production by metabolically engineered escherichia coli
WO2014004753A1 (en) * 2012-06-29 2014-01-03 Archer Daniels Midland Company Betaine enhancement of fermentation to make c4 diacids
US20150147792A1 (en) * 2012-06-29 2015-05-28 Archer Daniels Midland Company Betaine Enhancement of Fermentation to make C4 Diacids
WO2014018902A2 (en) * 2012-07-26 2014-01-30 Joule Unlimited Technologies, Inc. Methods and compositions for the augmentation of pyruvate and acetyl-coa formation
WO2014018902A3 (en) * 2012-07-26 2014-04-03 Joule Unlimited Technologies, Inc. Methods and compositions for the augmentation of pyruvate and acetyl-coa formation
US10465213B2 (en) 2012-08-10 2019-11-05 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
US10815473B2 (en) 2013-03-15 2020-10-27 Cargill, Incorporated Acetyl-CoA carboxylases
US10155937B2 (en) * 2013-03-15 2018-12-18 Cargill, Incorporated Acetyl-CoA carboxylases
US10047383B2 (en) 2013-03-15 2018-08-14 Cargill, Incorporated Bioproduction of chemicals
US20170101638A1 (en) * 2013-03-15 2017-04-13 Cargill, Incorporated Acetyl-coa carboxylases
US10006063B2 (en) * 2013-05-24 2018-06-26 Tianjin Institute Of Industrial Biotechnology, Chinese Academy Of Sciences Recombinant Escherichia coli for producing succinate acid and application thereof
EP3006556A4 (en) * 2013-05-24 2016-11-30 Tianjin Inst Ind Biotechnology Cas ESCHERICHIA COLI RECOMBINANT FOR PRODUCING SUCCINIC ACID AND CORRESPONDING APPLICATION
US20160097064A1 (en) * 2013-05-24 2016-04-07 Tianjin Institute Of Industrial Biotechnology, Chinese Academy Of Sciences Recombinant escherichia coli for producing succinic acid and application thereof
US11408013B2 (en) 2013-07-19 2022-08-09 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
US10337038B2 (en) 2013-07-19 2019-07-02 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
WO2015013334A1 (en) * 2013-07-23 2015-01-29 Myriant Corporation Method of producing succinic acid and other chemicals using facilitated diffusion for sugar import
US10494654B2 (en) 2014-09-02 2019-12-03 Cargill, Incorporated Production of fatty acids esters
US10301653B2 (en) * 2015-07-06 2019-05-28 Wisconsin Alumni Research Foundation Microorganisms that co-consume glucose with non-glucose carbohydrates and methods of use
US10982234B2 (en) 2015-07-06 2021-04-20 Wisconsin Alumni Research Foundation Microorganisms that co-consume glucose with non-glucose carbohydrates and methods of use
CN105062942A (zh) * 2015-08-26 2015-11-18 逢甲大学 可生产正丁醇的菌株及其应用
US9970016B2 (en) 2015-11-12 2018-05-15 Industrial Technology Research Institute Genetic engineered bacteria and methods for promoting production of succinic acid or lactic acid
US11345938B2 (en) 2017-02-02 2022-05-31 Cargill, Incorporated Genetically modified cells that produce C6-C10 fatty acid derivatives
CN113278567A (zh) * 2020-02-20 2021-08-20 杭州立丞生物科技有限公司 可改善菌株以提升生产重组蛋白质的方法
CN111411062A (zh) * 2020-05-07 2020-07-14 广东省农业科学院植物保护研究所 一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用
CN112300968A (zh) * 2020-11-19 2021-02-02 河南巨龙生物工程股份有限公司 产环磷腺苷的节杆菌及其应用

Also Published As

Publication number Publication date
MX2011010373A (es) 2011-10-12
AU2010232533A1 (en) 2011-10-13
US20150284746A1 (en) 2015-10-08
CN102803470A (zh) 2012-11-28
KR101711308B1 (ko) 2017-02-28
MX337341B (es) 2016-02-26
AU2016201951A1 (en) 2016-04-21
JP2012522515A (ja) 2012-09-27
BRPI1009992A2 (pt) 2020-08-18
AU2016201951B2 (en) 2017-12-21
SG175002A1 (en) 2011-11-28
EP2414505A4 (en) 2013-10-09
CA2756705A1 (en) 2010-10-07
CA2756705C (en) 2017-04-11
JP6072130B2 (ja) 2017-02-01
JP2015164429A (ja) 2015-09-17
KR20150018902A (ko) 2015-02-24
US10287611B2 (en) 2019-05-14
WO2010115067A3 (en) 2011-03-10
EP2414505B1 (en) 2019-05-22
EP2414505A2 (en) 2012-02-08
KR20120025450A (ko) 2012-03-15
WO2010115067A2 (en) 2010-10-07

Similar Documents

Publication Publication Date Title
US10287611B2 (en) Engineering the pathway for succinate production
US8691539B2 (en) Materials and methods for efficient succinate and malate production
Jantama et al. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate
US8778656B2 (en) Organic acid production in microorganisms by combined reductive and oxidative tricaboxylic acid cylce pathways
Jantama Metabolic engineering of Escherichia coli to efficiently produce succinate in mineral salts media

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC., F

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ZHANG, XUELI;JANTAMA, KAEMWICH;MOORE, JONATHAN C.;AND OTHERS;SIGNING DATES FROM 20110928 TO 20111020;REEL/FRAME:027160/0080

AS Assignment

Owner name: ENERGY, UNITED STATES DEPARTMENT OF, DISTRICT OF C

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF FLORIDA;REEL/FRAME:029236/0470

Effective date: 20111130

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION