Microorganisms and methods for improving product yields on methanol using acetyl-CoA synthesis

Patent metadata

Field	Value
Publication number	US-12529078-B2
Application number	US-202318390570-A
Country	US
Kind code	B2
Filing date	Dec 20, 2023
Priority date	Dec 3, 2013
Publication date	Jan 20, 2026
Grant date	Jan 20, 2026

Abstract

The invention provides non-naturally occurring microbial organisms containing enzymatic pathways and/or metabolic modifications for enhancing carbon flux through acetyl-CoA. In some embodiments, the microbial organisms of the invention having such pathways also include pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation. The enhanced carbon flux through acetyl-CoA, in combination with pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation can, in some embodiments, be used for production of a bioderived compound. Accordingly, in some embodiments, the microbial organisms of the invention can include a pathway capable of producing a bioderived compound of the invention. The invention still further provides a bioderived compound produced by a microbial organism of the invention, culture medium having the bioderived compound of the invention, compositions having the bioderived compound of the invention, a biobased product comprising the bioderived compound of the invention, and a process for producing a bioderived compound of the invention.

First claim

What is claimed is:

1 . A non-naturally occurring microbial organism having a methanol metabolic pathway, an acetyl-CoA pathway, and a formate assimilation pathway wherein said methanol metabolic pathway comprises a methanol dehydrogenase or a methanol methyltransferase, wherein said methanol dehydrogenase catalyzes conversion of methanol to formaldehyde and wherein said methanol methyltransferase catalyzes conversion of methanol into methyl-THF,
wherein said acetyl-CoA pathway comprises a xylulose-5-phosphate phosphoketolase wherein said xylulose-5-phosphate phosphoketolase catalyzes conversion of xylulose-5-phosphate (Xu5P) and phosphate to acetyl-phosphate (ACTP) and glyceraldehyde-3-phosphate (G3P),
wherein said formate assimilation pathway comprises:
(1) a pyruvate formate lyase, wherein said pyruvate formate lyase catalyzes conversion of pyruvate to acetyl-CoA (ACCOA); or
(2) a pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, or pyruvate: NADP+ oxidoreductase, and a formate dehydrogenase, wherein said pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, or pyruvate: NADP+ oxidoreductase catalyzes conversion of pyruvate to acetyl-CoA (ACCOA), and where said formate dehydrogenase converts CO 2 to formate;
wherein an enzyme of the methanol metabolic pathway, an enzyme of the acetyl-CoA pathway, and at least one enzyme of the formate assimilation pathway is encoded by an exogenous nucleic acid and is expressed in a sufficient amount to increase the yield of acetyl-CoA per mole of methanol if compared to the yield of acetyl-CoA per mole of methanol in the absence of said enzymes.

2 . The non-naturally occurring microbial organism of claim 1 , wherein said formate assimilation pathway further comprises a glyceraldehyde-3-phosphate dehydrogenase or an enzyme of lower glycolysis encoded by an exogenous nucleic acid.

3 . The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism further comprises a formaldehyde fixation pathway, wherein said formaldehyde fixation pathway further comprises:
(1) a dihydroxyacetone synthase and a fructose-6-phosphate aldolase, wherein said dihydroxyacetone synthase transfers a glycoaldehyde group from Xu5P to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) and wherein said fructose-6-phosphate aldolase catalyzes conversion of DHA and G3P to fructose-6-phosphate; (2) a dihydroxyacetone synthase, wherein said dihydroxyacetone synthase transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P); or (3) a 3-hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase, wherein said 3-hexulose-6-phosphate synthase catalyzes conversion of formaldehyde and D-ribulose-5-phosphate to hexulose-6-phosphate and wherein said 6-phospho-3-hexuloisomerase catalyzes conversion of H6P to fructose-6-phosphate (F6P),
wherein an enzyme of the formaldehyde fixation pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to increase yield of acetyl-CoA per mole of methanol if compared to yield of acetyl-CoA per mole of methanol in the absence of said enzyme.

4 . The non-naturally occurring microbial organism of claim 1 , wherein said formate assimilation pathway further comprises:
(1) a formate reductase, wherein said formate reductase catalyzes conversion of formate to formaldehyde; (2) a formate ligase, and a formyl-CoA reductase, wherein said formate ligase catalyzes conversion of formate to formyl-CoA and wherein said formyl-CoA reductase catalyzes conversion of formyl-CoA to formaldehyde; (3) a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a formaldehyde-forming enzyme or spontaneous, wherein said formyltetrahydrofolate synthetase catalyzes conversion of formate to formyltetrahydrofolate, wherein said methenyltetrahydrofolate cyclohydrolase catalyzes conversion of formyltetrahydrofolate to methenyltetrahydrofolate, wherein said methylenetetrahydrofolate dehydrogenase catalyzes conversion of methenyltetrahydrofolate to methylenetetrahydrofolate, wherein said formaldehyde-forming enzyme catalyzes conversion of methylenetetrahydrofolate to formaldehyde and tetrahydrofolate or wherein said conversion of methylenetetrahydrofolate to formaldehyde and tetrahydrofolate is spontaneous; (4) a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, a glycine cleavage system, a serine hydroxymethyltransferase, and a serine deaminase, wherein said formyltetrahydrofolate synthetase catalyzes conversion of formate to formyltetrahydrofolate, wherein said methenyltetrahydrofolate cyclohydrolase catalyzes conversion of formyltetrahydrofolate to methenyltetrahydrofolate, wherein said methylenetetrahydrofolate dehydrogenase catalyzes conversion of methenyltetrahydrofolate to methylenetetrahydrofolate, wherein said glycine cleavage system catalyzes transfer a formaldehyde group from methylenetetrahydrofolate to glycine, wherein said serine hydroxymethyltransferase catalyzes conversion of glycine to serine and wherein said serine deaminase catalyzes conversion of serine to pyruvate; (5) a formate reductase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, a glycine cleavage system, a serine hydroxymethyltransferase, and a serine deaminase, wherein said formate reductase catalyzes conversion of formate to formaldehyde, wherein said formyltetrahydrofolate synthetase catalyzes conversion of formate to formyltetrahydrofolate, wherein said methenyltetrahydrofolate cyclohydrolase catalyzes conversion of formyltetrahydrofolate to methenyltetrahydrofolate, wherein said methylenetetrahydrofolate dehydrogenase catalyzes conversion of methenyltetrahydrofolate to methylenetetrahydrofolate, wherein said glycine cleavage system catalyzes transfer of a formaldehyde group from methylenetetrahydrofolate to glycine, wherein said serine hydroxymethyltransferase catalyzes conversion of glycine to serine and wherein said serine deaminase catalyzes conversion of serine to pyruvate; (6) a formate ligase, a formyl-CoA reductase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, a glycine cleavage system, a serine hydroxymethyltransferase, and a serine deaminase, wherein said formate ligase catalyzes conversion of formate to formyl-CoA, wherein said formyl-CoA reductase catalyzes conversion of formyl-CoA to formaldehyde, wherein said formyltetrahydrofolate synthetase catalyzes conversion of formate to formyltetrahydrofolate, wherein said methenyltetrahydrofolate cyclohydrolase catalyzes conversion of formyltetrahydrofolate to methenyltetrahydrofolate, wherein said methylenetetrahydrofolate dehydrogenase catalyzes conversion of methenyltetrahydrofolate to methylenetetrahydrofolate, wherein said glycine cleavage system catalyzes transfer of a formaldehyde group from methylenetetrahydrofolate to glycine, wherein said serine hydroxymethyltransferase catalyzes conversion of glycine to serine and wherein said serine deaminase catalyzes conversion of serine to pyruvate; (7) a formaldehyde-forming enzyme or spontaneous, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, a glycine cleavage system, a serine hydroxymethyltransferase, and a serine deaminase, wherein said formaldehyde-forming enzyme catalyzes conversion of methylenetetrahydrofolate to formaldehyde and tetrahydrofolate or wherein said conversion of methylenetetrahydrofolate to f

Assignees

• Genomatica Inc

Inventors

• Osterhout Robin E
• Burgard Anthony P
• Pharkya Priti
• Andrae Stefan

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12P13/005

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P7/04

  acyclic · CPC title