Engineering the pathway for succinate production

Abstract

This invention relates to the biocatalysts for the efficient production of succinic acid and/or other products from renewable biological feedstocks. The biocatalysts have a very high efficiency for the growth-coupled production of succinic acid and/or other products from carbohydrate feed stocks as a result of both genetic manipulations and metabolic evolution. More specifically, certain biocatalysts of the present invention produce succinic acid at high titers and yield in mineral salts media during simple pH-controlled, batch fermentation without the addition of any exogenous genetic material. The genetic manipulations of the present invention are concerned with the energy-conserving strategies coupled with the elimination of alternative routes for NADH oxidation other than the routes for succinic acid production. The biocatalysts contain glucose-repressed gluconeogenic phosphoenol pyruvate carboxykinase (pck) depressed by genetic modifications and a genetically-inactivated phosphotransferase system. In terms of succinic acid production efficiency, the biocatalysts of the present invention are functionally equivalent to succinate producing rumen bacteria such as Actinobacillus succinogens and Mannheimia succiniproducens with one difference that the biocatalysts are able to achieve this high level of succinic acid production in a minimal salt medium with carbohydrate source as opposed to the requirement for a rich media for succinic acid production by rumen bacteria.

1 - 44 . (canceled) 
     
     
         45 . An isolated bacterial cell having increased phosphoenol pyruvate carboxykinase (PCK) activity. 
     
     
         46 . The bacterial cell of  claim 45 , wherein said bacterial cell is a non-ruminant bacterial cell. 
     
     
         47 . The bacterial strain of  claim 45 , wherein said bacterium is  Escherichia coli, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus  sp. CCM825,  Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus  sp. ATCC 15592,  Rhodococcus  sp. ATCC 19070 , Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquifaciens, Bacillus coagulans, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri , or  Xanthomonas citri.    
     
     
         48 . The bacterial cell of  claim 45 , wherein the increase in pck activity results from increased levels of pck transcripts within the cell. 
     
     
         49 . The bacterial cell of  claim 48 , wherein said increased levels of pck transcripts result from replacement of native regulatory sequences with altered regulatory sequences of the pck gene that increase pck transcription or with exogenous promoter sequencesr. 
     
     
         50 . The bacterial cell of  claim 45 , wherein the increase in pck activity results from increased levels of pck transcripts result arising from one or more mutations in the promoter region of the pck gene. 
     
     
         51 . The bacterial cell of  claim 50 , wherein said one or more mutations are point mutations comprising replacement of nucleotide A with nucleotide G at position 68 up stream of the pck gene start codon. 
     
     
         52 . The bacterial cell of  claim 45 , further comprising one or more genetic modification that disrupts the functioning of PEP-dependent phosphotransferase system. 
     
     
         53 . The bacterial cell of  claim 52 , wherein said genetic modification is in one or more genes coding for the structural components of PEP-dependent phosphotransferase system. 
     
     
         54 . The bacterial cell of  claim 52 , wherein said genetic modification is in one or more genes coding for proteins that regulate the expression of PEP-dependent phosphotransferase system. 
     
     
         55 . The bacterial cell of  claim 52 , wherein the said genetic modification is in one or more genes selected from the group consisting of ptsG, ptsH, ptsI, crr and crp. 
     
     
         56 . The bacterial cell of  claim 45 , further comprising: (a) one or more genetic modification that disrupts the functioning of PEP-dependent phosphotransferase system; and (b) one or more genetic modifications that upregulate the expression of one or more genes encoding sugar transporters. 
     
     
         57 . The bacterial cell of  claim 45 , further comprising genetic modification leading to the inactivation of gene expression in one or more genes involved in the fermentative pathway. 
     
     
         58 . The bacterial cell of  claim 45 , further comprising genetic modification leading to the inactivation of gene expression in one or more genes selected from a group consisting of adhE, ldhA, focA, pflA, ack, pta, pdh, mgsA, tdcD, tdcE and poxB. 
     
     
         59 . The bacterial cell of  claim 45 , further comprising: (a) one or more genetic modification that disrupts the functioning of PEP-dependent phosphotransferase system; and (b) mutation in one or more genes involved in the fermentative pathway. 
     
     
         60 . The bacterial cell of  claim 45 , further comprising: (a) genetic modification in one or more genes selected from a group consisting of ptsG, ptsH, ptsI, crr and crp; and (b) genetic modifications leading to the inactivation of gene expression in one or more genes selected from a group consisting of adhE, ldhA, focA, pflA, ack, pta, pdh, mgsA, tdcD, tdcE and poxB. 
     
     
         61 . The bacterial cell of  claim 45 , further comprising genetic modification in one or more genes associated with the operation of the TCA cycle. 
     
     
         62 . The bacterial cell of  claim 45 , further comprising genetic modification in one or more genes selected from a group consisting of mdh, fumA, fumB, fumC, frdABCD, acceAB, acnAB, icd, iclR, aspC, and scfA. 
     
     
         63 . The bacterial cell of  claim 45 , further comprising: (a) genetic modification in one or more genes selected from a group consisting of ptsG, ptsH, ptsI, crr and crp; (b) genetic modifications leading to the inactivation of gene expression in one or more genes selected from a group consisting of adhE, ldhA, focA, pflA, ack, pta, pdh, mgsA, tdcD, tdcE and poxB; and (c) genetic modification in one or more genes selected from a group consisting of mdh, fumA, fumB, fumC, frdABCD, acceAB, acnAB, icd, iclR, aspC, and scfA. 
     
     
         64 . The bacterial strain of  claim 45 , further comprising genetic modification in one or more genes selected from a group consisting of phosphoenol pyruvate carboxylase, NADH dependent malic enzyme and NADPH dependent malic enzyme. 
     
     
         65 . The bacterial strain of  claim 45 , further comprising an exogenous pyruvate carboxylase. 
     
     
         66 . A genetically modified bacterial cell, wherein said genetically modified bacterial cell is XZ320, XZ332, XZ341, XZ468, XZ469, XZ470, XZ613, XZ615, XZ616, XZ618, XZ620, XZ647, XZ721, or XZ723. 
     
     
         67 . An  Escherichia coli  bacterial strain comprising: (a) an inactivated ldhA; (b) inactivated focA; (c) an inactivated pflB; (d) inactivated ackA; (e) an inactivated mgsA; (f) an inactivated adhE; (g) an inactivated tdcD; (h) an inactivated tdcE; (i) an inactivated aspC; (j) an inactivated sfcA; (k) an inactivated ptsH; (l) an inactivated citD and (in) an up regulated pck. 
     
     
         68 . A method of producing succinic acid comprising:
 a) culturing a bacterial strain of  claim 45 ;   b) providing a carbon source;   c) allowing said bacteria to metabolize said carbon source; and   d) isolating succinic acid.