Method for producing objective substance

Patent metadata

Field	Value
Publication number	US-11680279-B2
Application number	US-201816200011-A
Country	US
Kind code	B2
Filing date	Nov 26, 2018
Priority date	Nov 29, 2017
Publication date	Jun 20, 2023
Grant date	Jun 20, 2023

Abstract

A method for producing an objective substance such as vanillin and vanillic acid is provided. An objective substance is produced from a carbon source or a precursor of the objective substance by using a microorganism that is able to produce the objective substance, which microorganism has been modified so that the activity of an enzyme involved in SAM cycle (SAM cycle enzyme) is increased.

First claim

The invention claimed is:

1. A method for producing an objective substance, the method comprising:
producing the objective substance by using a microorganism having an ability to produce the objective substance,
wherein the microorganism has been modified so that the activity of an S-adenosylmethionine cycle enzyme is increased as compared with a non-modified microorganism,
wherein the microorganism is a coryneform bacterium,
wherein the S-adenosylmethionine cycle enzyme is a combination of methionine adenosyltransferase and 5,10-methylenetetrahydrofolate reductase, and optionally may include methionine synthase; and
wherein the objective substance is selected from
metabolites the biosynthesis of which requires S-adenosylmethionine selected from the group consisting of vanillin, vanillic acid, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, creatine, and combinations thereof;
wherein said producing comprises:
cultivating the microorganism in a culture medium comprising a carbon source so that the objective substance is produced and accumulates in the culture medium, or
converting a precursor of the objective substance into the objective substance by:
cultivating the microorganism in a culture medium comprising the precursor so that the objective substance is produced and accumulates in the culture medium, or
allowing cells of the microorganism to act on the precursor in a reaction mixture so that the objective substance is produced and accumulates in the reaction mixture.

2. The method according to claim 1 , wherein the cells are present in a culture broth of the microorganism, collected from a culture broth of the microorganism, present in a processed product of a culture broth of the microorganism, present in a processed product of the cells collected from a culture broth of the microorganism, or a combination thereof.

3. The method according to claim 1 , wherein the precursor is selected from the group consisting of protocatechuic acid, protocatechualdehyde, L-tryptophan, L-histidine, L-phenylalanine, L-tyrosine, L-arginine, L-ornithine, glycine, and combinations thereof.

4. The method according to claim 1 , the method further comprising collecting the objective substance.

5. The method according to claim 1 , wherein the methionine adenosyltransferase is encoded by an metK gene, the methionine synthase is encoded by an metH gene and/or an metE gene, and the 5,10-methylenetetrahydrofolate reductase is encoded by an metF gene.

6. The method according to claim 5 , wherein:
the metK gene encodes a protein selected from the group consisting of:

(1a) a protein comprising the amino acid sequence of SEQ ID NO: 162,
(1b) a protein comprising the amino acid sequence of SEQ ID NO: 162 that includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has methionine adenosyltransferase activity, and
(1c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 162, and wherein said protein has methionine adenosyltransferase activity;
the metH gene encodes a protein selected from the group consisting of:

(2a) a protein comprising the amino acid sequence of SEQ ID NO: 168 or 170,
(2b) a protein comprising the amino acid sequence of SEQ ID NO: 168 or 170 that includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has methionine synthase activity, and
(2c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 168 or 170, and wherein said protein has methionine synthase activity;
the metE gene encodes a protein selected from the group consisting of:

(3a) a protein comprising the amino acid sequence of SEQ ID NO: 166,
(3b) a protein comprising the amino acid sequence of SEQ ID NO: 166 that includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has methionine synthase activity, and
(3c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 166, and wherein said protein has methionine synthase activity; and
the metF gene encodes a protein selected from the group consisting of:

(4a) a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, or 184,
(4b) a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, or 184 that includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has 5,10-methylenetetrahydrofolate reductase activity, and
(4c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 172, 174, or 184, and wherein said protein has 5,10-methylenetetrahydrofolate reductase activity.

7. The method according to claim 1 , wherein the activity of the S-adenosylmethionine cycle enzyme is increased by increasing the expression of a gene encoding the S-adenosylmethionine cycle enzyme.

8. The method according to claim 7 , wherein the expression of the gene encoding the S-adenosylmethionine cycle enzyme is increased by increasing the copy number of the gene and/or modifying an expression control sequence of the gene.

9. The method according to claim 1 , wherein the microorganism is a bacterium belonging to the genus Corynebacterium.

10. The method according to claim 9 , wherein the microorganism is Corynebacterium glutamicum.

11. The method according to claim 1 , wherein the microorganism has been further modified so that the activity of an enzyme that is involved in the biosynthesis of the objective substance is increased as compared with a non-modified microorganism.

12. The method according to claim 11 , wherein the enzyme that is involved in the biosynthesis of the objective substance is selected from the group consisting of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, 3-dehydroshikimate dehydratase, O-methyltransferase, aromatic aldehyde oxidoreductase, and combinations thereof.

13. The method according to claim 1 , wherein the microorganism has been further modified so that the activity of phosphopantetheinyl transferase is increased as compared with a non-modified microorganism.

14. The method according to claim 1 , wherein the microorganism has been further modified so that the activity of an enzyme that is involved in the by-production of a substance other than the objective substance is reduced as compared with a non-modified microorganism.

15. The method according to claim 14 , wherein the enzyme that is involved in the by-production of a substance other than the objective substance is selected from the group consisting of vanillate demethylase, protocatechuate 3,4-dioxygenase, alcohol dehydrogenase, shikimate dehydrogenase, and combinations thereof.

16. The method according to claim 1 , wherein the microorganism has been further modified so that the activity of an L-cysteine biosynthesis enzyme is increased as compared with a non-modified microorganism.

17. The method according to claim 16 , wherein the L-cysteine biosynthesis enzyme is selected from the group consisting of proteins encoded by a cysI gene, cysX gene, cysH gene, cysD gene, cysN gene, cysY gene, cysZ gene, fpr2 gene, and combinations thereof.

18. The method according to

Assignees

• Ajinomoto Kk

Inventors

• Mijts Benjamin
• Roche Christine
• Murali Rekha
• Asari Sayaka
• Fukui Keita
• Toyazaki Miku
• Noguchi Keiko

Classifications

• C12R2001/19

  Escherichia coli · CPC title

• C12Y205/01006

  Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase · CPC title

• C12N9/1014

  Hydroxymethyl-, formyl-transferases (2.1.2) · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12R2001/46

  Streptococcus {; Enterococcus; Lactococcus} · CPC title