IP Library Granted Patent US 12,435,346
Granted Patent B2
US 12,435,346 · App. 18/453,987 · Granted Oct 7, 2025

Methods and organisms with increased carbon flux efficiencies

Inventors: Anthony P. Burgard (Elizabeth, PA); Robin E. Osterhout (San Diego, CA); Stephen J. Van Dien (Encinitas, CA); Priti Pharkya (San Diego, CA); Tae Hoon Yang (Encinitas, CA); Jungik Choi (San Diego, CA)
Assignee: Genomatica, Inc.
C12P7/18C07K14/245C12N1/20C12N9/0036C12N9/0057C12N9/1205C12N9/16C12N9/52C12N9/88C12N9/93C12N15/52C12P7/42C12Y106/01002C12Y110/03C12Y207/0104C12Y301/00C12Y301/02C12Y301/02002C12Y304/21092C12Y401/01031C12Y401/01032
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Quick Facts
Patent No.
US 12,435,346
App. No.
18/453,987
Granted
Oct 7, 2025
Kind
B2
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.

Claims (31)

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).

Assignments (3)
SECURITY INTEREST Recorded Feb 10, 2026
From: GENOMATICA, INC.
To: AGAIN BIO APS
Reel/Frame 074708/0001 →
SECURITY INTEREST Recorded Dec 9, 2025
From: GENOMATICA, INC.
To: NOVO HOLDINGS A/S, AS COLLATERAL AGENT
Reel/Frame 073915/0027 →
SECURITY INTEREST Recorded Jun 2, 2025
From: GENOMATICA, INC.
To: OXFORD FINANCE LLC
Reel/Frame 071471/0770 →
Continuity (5)
Continuation 16785510 · Feb 7, 2020
Continuation 15108259
Provisional Application 62013390 · Jun 17, 2014
Provisional Application 61921292 · Dec 27, 2013
Related Publication 20240124904A1 · Apr 18, 2024
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