Methods and organisms with increased carbon flux efficiencies
The invention is directed to a non-naturally occurring microbial organism comprising a first attenuation of a succinyl-CoA synthetase or transferase and at least a second attenuation of a succinyl-CoA converting enzyme or a gene encoding a succinate producing enzyme within a multi-step pathway having a net conversion of succinyl-CoA to succinate.
1. A non-naturally occurring microbial organism comprising a gene disruption of a gene encoding YciA CoA hydrolase, increased expression of a pyridine nucleotide transhydrogenase, and a metabolically engineered pathway for producing a bioderived compound from a TCA cycle intermediate;
wherein the microbial organism further comprises one or more modifications selected from the group consisting of:
(a) a genetic alteration that increases expression of NADH dehydrogenase Ndh-I, cytochrome bo oxidase, or both NADH dehyrogenase Ndh-I and cytochrome bo oxidase;
(b) attenuation of one or more NAD (P) H dehydrogenases or NAD (P) H: quinine oxidoreductases, one or more ubiquinol oxidases, one or more NAD (P) H dehydrogenases, or one or more ubiquinol oxidases;
(c) a gene disruption of one or more endogenous nucleic acids encoding a menaquinol biosynthetic enzyme, or one or more nucleic acids encoding a dimethylmenaquinol biosynthetic enzyme;
(d) attenuation of protein encoding ATP-dependent Clp protease ATP-binding subunit (ClpA), pyruvate kinase or glucose phosphotransferase system (PTS); and
(e) a genetic alteration that increases expression of a phosphoenoylpyruvate carboxykinase (PEPCK), a phosphoenoylpyruvate carboxylase (PPC), or both a PEPCK and a PPC in said microbial organism.
2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is selected from the group consisting of bacteria, yeast, fungus or other microorganism applicable to a fermentation process.
3. A method of producing a bioderived compound comprising culturing a non-naturally occurring microbial organism of claim 1 for a sufficient period of time under conditions sufficient to produce said bioderived compound.
4. The non-naturally occurring microbial organism of claim 1 , wherein:
(i) said increased expression of a pyridine nucleotide transhydrogenase comprises up regulating the expression of an endogenous pyridine nucleotide transhydrogenase;
(ii) said pyridine nucleotide transhydrogenase comprises a proton-translocating transhydrogenase; or
iii) said pyridine nucleotide transhydrogenase is encoded by pntAB.
5. The non-naturally occurring microbial organism of claim 1 , further comprising attenuation of a TCA cycle enzyme other than a succinyl-CoA synthetase or transferase.
6. The non-naturally occurring microbial organism of claim 5 , wherein said TCA cycle enzyme other than a succinyl-CoA synthetase or transferase is selected from succinic dehydrogenase, fumarase and malate dehydrogenase.
7. The non-naturally occurring microbial organism of claim 1 , wherein said bioderived compound is selected from the group consisting of 4-hydroxybutyrate (4HB), 1,4-butanediol (1,4-BDO), 1,3-butanediol (1,3-BDO), polyhydroxylbutanoate (PHB), butadiene, adipate, 6-aminocaproate, caprolactam, methacrylic acid, isopropanol, long chain alcohols, hexamethylenediamene, methyl methacrylate, butanol, 3-butene-1-ol, 3-butene-2-ol and crotyl-alcohol.
8. The non-naturally occurring microbial organism of claim 7 , wherein said bioderived compound is 4HB, 1,4-BDO, or 1,3-BDO.
9. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the genetic alteration that increases expression of NADH dehyrogenase Ndh-I, cytochrome bo oxidase, or both NADH dehyrogenase Ndh-I and cytochrome bo oxidase.
10. The non-naturally occurring microbial organism of claim 9 , wherein said microbial organism comprises an exogenous nucleic acid encoding said NADH dehyrogenase Ndh-I (nuo), cytochrome bo oxidase (cyoABCDE) or both NADH dehyrogenase Ndh-I (nuo) and cytochrome bo oxidase (cyoABCDE).
11. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the attenuation of one or more NAD (P) H dehydrogenases or NAD (P) H: quinine oxidoreductases, one or more ubiquinol oxidases, or both one or more NAD (P) H dehydrogenases and one or more ubiquinol oxidases;
wherein the one or more NAD (P) H dehydrogenases or NAD (P) H: quinine oxidoreductases are selected from the group consisting of Ndh-II, WrbA, YhdH, YieF, YtfG, Qor and MdaB, the one or more ubiquinol oxidases are selected from the group consisting of cytochrome bd-I oxidase, cytochrome bd-II oxidase and quinol monooxygenase, the one or more NAD (P) H dehydrogenases are selected from the group consisting of Ndh-II, WrbA, YhdH, YieF, YtfG, Qor and MdaB, and the one or more ubiquinol oxidases are selected from the group consisting of cytochrome bd-I oxidase, cytochrome bd-II oxidase and quinol monooxygenase.
12. The non-naturally occurring microbial organism of claim 11 , wherein said attenuation comprises attenuating said Ndh-II, bd-I oxidase or Ndh-II and bd-I oxidase.
13. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the gene disruption of one or more endogenous nucleic acids encoding a menaquinol biosynthetic enzyme or one or more nucleic acids encoding a dimethylmenaquinol biosynthetic enzyme.
14. The non-naturally occurring microbial organism of claim 13 , wherein one or more endogenous nucleic acids encoding a menaquinol or dimethylmenaquinol biosynthetic enzyme is a nucleic acid encoding a menF, menD, menH, menC, menE, menB, menI, menA or menG gene, or an ortholog having at least 70% identity to a menF, menD, menH, menC, menE, menB, menI, menA or menG gene.
15. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the attenuation of protein encoding ClpA, pyruvate kinase or glucose phosphotransferase system (PTS).
16. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the genetic alteration that increases expression of a phosphoenoylpyruvate carboxykinase (PEPCK) in said microbial organism.
17. The non-naturally occurring microbial organism of claim 16 , wherein:
(i) said microbial organism comprises an exogenous nucleic acid encoding said PEPCK; or
(ii) said microbial organism further comprises a genetic alteration that comprises attenuation of a phosphoenoylpyruvate carboxylase (PPC) in said microbial organism.
18. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises the genetic alteration that increases expression of a phosphoenoylpyruvate carboxykinase (PEPCK), phosphenolpyruvate carboxylase (PPC), or both a PEPCK and a PPC in said microbial organism.
19. The non-naturally occurring microbial organism of claim 18 , further comprising attenuation of a pyruvate kinase or glucose phosphotransferase system (PTS).