Method for producing objective substance

Patent metadata

Field	Value
Publication number	US-10876138-B2
Application number	US-201916392791-A
Country	US
Kind code	B2
Filing date	Apr 24, 2019
Priority date	Oct 26, 2016
Publication date	Dec 29, 2020
Grant date	Dec 29, 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 that the activity of enolase is reduced.

First claim

The invention claimed is:

1. A method for producing an objective substance, the method comprising the following step:
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 enolase is reduced as compared with a non-modified microorganism, and
wherein the objective substance is selected from the group consisting of vanillin, vanillic acid, melatonin, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, creatine, L-methionine, and combinations thereof
wherein said producing comprises:
A) cultivating the microorganism in a culture medium containing a carbon source to produce and accumulate the objective substance in the culture medium, or
B) converting a precursor of the objective substance into the objective substance by:
i) cultivating the microorganism in a culture medium containing the precursor to produce and accumulate the objective substance in the culture medium, or
ii) allowing cells of the microorganism to act on the precursor in a reaction mixture to produce and accumulate the objective substance in the reaction mixture.

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

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 enolase is a protein encoded by eno gene.

6. The method according to claim 5 , wherein the eno gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 129,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 129 but that includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has enolase 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: 129, and wherein said protein has enolase activity.

7. The method according to claim 1 , wherein the activity of enolase is reduced by attenuating the expression of a gene encoding enolase, or by disrupting a gene encoding enolase.

8. The method according to claim 7 , wherein the expression of the gene encoding enolase is attenuated by modifying an expression control sequence of the gene.

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

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

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

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

13. The method according to claim 12 , wherein the microorganism is Escherichia coli.

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 biosynthesis of the objective substance is increased as compared with a non-modified microorganism.

15. The method according to claim 14 , 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.

16. 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.

17. 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.

18. The method according to claim 17 , 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.

19. 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.

20. The method according to claim 19 , wherein the L-cysteine biosynthesis enzyme is a protein encoded by a gene selected from the group consisting of cysl gene, cysX gene, cysH gene, cysD gene, cysN gene, cysY gene, cysZ gene, fpr2 gene, and combinations thereof.

21. The method according to claim 19 , wherein the activity of the L-cysteine biosynthesis enzyme is increased by increasing the activity of a protein encoded by cysR gene.

22. The method according to claim 1 , wherein the microorganism has been further modified so that the activity of a protein encoded by NCg12048 gene is reduced as compared with a non-modified microorganism.

23. A method for producing vanillin, the method comprising:
producing vanillic acid by the method according to claim 1 ; and
converting said vanillic acid to vanillin.

24. The method according to claim 23 , wherein the microorganism is a bacterium belonging to the genus Corynebacterium.

25. The method according to claim 23 , wherein the microorganism is Corynebacterium glutamicum.

Assignees

• Ajinomoto Kk

Inventors

• Mijts Benjamin
• Roche Christine
• Asari Sayaka
• Toyazaki Miku
• Fukui Keita

Classifications

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title

• C12R2001/15

  Corynebacterium · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12P13/12

  Methionine; Cysteine; Cystine · CPC title