Methods and organisms with increased carbon flux efficiencies

Patent metadata

Field	Value
Publication number	US-12435346-B2
Application number	US-202318453987-A
Country	US
Kind code	B2
Filing date	Aug 22, 2023
Priority date	Dec 27, 2013
Publication date	Oct 7, 2025
Grant date	Oct 7, 2025

Abstract

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.

First claim

What is claimed is:

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).

Assignees

• Genomatica Inc

Inventors

• Burgard Anthony P
• Osterhout Robin E
• Van Dien Stephen J
• Pharkya Priti
• Yang Tae Hoon
• Choi Jungik

Classifications

• C12Y401/01032

  Phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32) · CPC title

• C12Y401/01031

  Phosphoenolpyruvate carboxylase (4.1.1.31) · CPC title

• C12Y304/21092

  Endopeptidase Clp (3.4.21.92) · CPC title

• C12Y301/02002

  Palmitoyl-CoA hydrolase (3.1.2.2) · CPC title

• C12Y301/02

  Thioester hydrolases (3.1.2) · CPC title