Method for producing objective substance

Patent metadata

Field	Value
Publication number	US-10858676-B2
Application number	US-201815982623-A
Country	US
Kind code	B2
Filing date	May 17, 2018
Priority date	May 22, 2017
Publication date	Dec 8, 2020
Grant date	Dec 8, 2020

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 having an objective substance-producing ability, which microorganism has been modified so as to have a specific feature, such as a reduced activity of AICAR formyltransferase/IMP cyclohydrolase, an increased activity of 3-PGDH, and/or a reduced activity of L-serine deaminase.

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 a feature selected from the group consisting of
(A) a first feature selected from the group consisting of
(A-i) the microorganism has been modified so that the activity of AICAR formyltransferase/IMP cyclohydrolase is reduced as compared with a non-modified microorganism,
(A-ii) the microorganism has been modified so that a gene encoding AICAR formyltransferase/IMP cyclohydrolase has a mutation that improves the ability of the microorganism to produce the objective substance, and
(A-iii) combinations thereof;

(B) a second feature selected from the group consisting of
(B-i) the microorganism has been modified so that the activity of D-3-phosphoglycerate dehydrogenase is increased as compared with a non-modified microorganism,
(B-ii) the microorganism has been modified so as to have a gene encoding D-3-phosphoglycerate dehydrogenase resistant to feedback inhibition by L-serine, and
(B-iii) combinations thereof;

(C) the microorganism has been modified so that the activity of L-serine deaminase is reduced as compared with a non-modified microorganism; and
(D) combinations thereof;
wherein the objective substance is a metabolite selected from the group consisting of vanillin, vanillic acid, melatonin, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, creatine, and combinations thereof.

2. The method according to claim 1 , 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.

3. The method according to claim 1 , wherein said producing comprises
converting a precursor of the objective substance into the objective substance by using the microorganism.

4. The method according to claim 3 , wherein said using comprises
cultivating the microorganism in a culture medium comprising the precursor so that the objective substance is produced and accumulates in the culture medium.

5. The method according to claim 3 , wherein said converting comprises
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.

6. The method according to claim 5 , 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 of these.

7. The method according to claim 3 , 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.

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

9. The method according to claim 1 , wherein the AICAR formyltransferase/IMP cyclohydrolase is encoded by a purH gene.

10. The method according to claim 9 , wherein the purH gene encodes a protein selected from the group consisting of
(a) a protein comprising the amino acid sequence of SEQ ID NO: 135,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 135 but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has AICAR formyltransferase/IMP cyclohydrolase activity, and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 135, and wherein said protein has AICAR formyltransferase/IMP cyclohydrolase activity.

11. The method according to claim 1 , wherein the activity of AICAR formyltransferase/IMP cyclohydrolase is reduced by a method selected from the group consisting of
(A) attenuating the expression of a gene encoding AICAR formyltransferase/IMP cyclohydrolase,
(B) disrupting a gene encoding AICAR formyltransferase/IMP cyclohydrolase,
(C) modifying a gene encoding AICAR formyltransferase/IMP cyclohydrolase so as to have a mutation that improves the ability of the microorganism to produce the objective substance, and
(D) combinations thereof.

12. The method according to claim 11 , wherein said attenuating or said disrupting comprises deletion of the gene encoding AICAR formyltransferase/IMP cyclohydrolase.

13. The method according to claim 1 , wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae, a coryneform bacterium, or yeast.

14. The method according to claim 13 , wherein the coryneform bacterium is a bacterium belonging to the genus Corynebacterium.

15. The method according to claim 14 , wherein the bacterium belonging to the genus Corynebacterium is Corynebacterium glutamicum.

16. The method according to claim 13 , wherein the bacterium belonging to the family Enterobacteriaceae is a bacterium belonging to the genus Escherichia.

17. The method according to claim 16 , wherein the bacterium belonging to the genus Escherichia is Escherichia coli.

18. 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.

19. The method according to claim 18 , wherein said enzyme 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.

20. 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.

21. 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.

22. The method according to claim 21 , wherein said enzyme is selected from the group consisting of vanillate demethylase, protocatechuate 3,4-dioxygenase, alcohol dehydrogenase, shikimate dehydrogenase, and combinations thereof.

23. 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.

24. The method according to claim 23 , wherein the L-cysteine biosynthesis enzyme is encoded by a gene selected from the group consisting of a cysI gene, cysX gene, cysH gene, cysD gene, cysN gene, cysY gene, cysZ gene, fpr2 gene, and combinations thereof.

25. The method according to claim 23 , wherein the activity of the L-cysteine biosynthesis enzyme is increased by increasing the activity of a protein encoded by a cysR gene.

26. The method according to claim 1 , wherein the microorganism has been further modified so that the activity

Assignees

• Ajinomoto Kk

Inventors

• Roche Christine
• Mijts Benjamin
• Kelly Peter
• Davis Matthew
• Asari Sayaka
• Fukui Keita
• Toyazaki Miku

Classifications

• C12R2001/15

  Corynebacterium · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12P7/24 Primary

  containing a carbonyl group · CPC title

• C12P7/22

  aromatic · CPC title