# US 2019/0249206 A1 · Method for Producing Objective Substance > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0249206 A1 - Document ID: US20190249206A1 - Status: Published application - Kind: A1 - Application number: 16/392,791 - Filed: 2019-04-24 - Published: 2019-08-15 - Earliest claimed: 2016-10-26 - Assignees: Ajinomoto Co., Inc. - Applicants: Ajinomoto Co., Inc. - Inventors: Benjamin Mijts; Christine Roche; Sayaka Asari; Miku Toyazaki; Keita Fukui - Web page: https://patentgrep.com/patent/US20190249206A1 ## CPC - C12P 7/24: containing a carbonyl group - C12N 1/205: Bacterial isolates - C12N 1/36: Adaptation or attenuation of cells - C12P 7/42: Hydroxy-carboxylic acids - C12P 13/12: Methionine; Cysteine; Cystine - C12N 2330/50: Biochemical production, i.e. in a transformed host cell - C12N 2510/02: Cells for production - C12R 2001/15: Corynebacterium - C12R 2001/19: Escherichia coli - C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1) - C12Y 101/01025: Shikimate dehydrogenase (1.1.1.25) - C12Y 113/11003: Protocatechuate 3,4-dioxygenase (1.13.11.3) - C12Y 114/13082: Vanillate monooxygenase (1.14.13.82) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS - C12Y: ENZYMES ## 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. ## Claims ### Claim 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: (X) metabolites the biosynthesis of which requires S-adenosylmethionine, (Y) L-methionine, and (Z) combinations thereof. ### Claim 2 (depends on 1) The method according to claim 1, wherein said producing comprises: cultivating the microorganism in a culture medium containing a carbon source to produce and accumulate the objective substance in the culture medium. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 3) The method according to claim 3, wherein said converting comprises: cultivating the microorganism in a culture medium containing the precursor to produce and accumulate the objective substance in the culture medium. ### Claim 5 (depends on 3) The method according to claim 3, wherein said converting comprises: 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. ### Claim 6 (depends on 5) The method according to claim 5, 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. ### Claim 7 (depends on 3) 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. ### Claim 8 (depends on 1) The method according to claim 1, the method further comprising collecting the objective substance. ### Claim 9 (depends on 1) The method according to claim 1, wherein the enolase is a protein encoded by eno gene. ### Claim 10 (depends on 9) The method according to claim 9, 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 11) The method according to claim 11, wherein the expression of the gene encoding enolase is attenuated by modifying an expression control sequence of the gene. ### Claim 13 (depends on 1) The method according to claim 1, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae, a coryneform bacterium, or yeast. ### Claim 14 (depends on 13) The method according to claim 13, wherein the microorganism is a bacterium belonging to the genus Corynebacterium. ### Claim 15 (depends on 14) The method according to claim 14, wherein the microorganism is Corynebacterium glutamicum. ### Claim 16 (depends on 13) The method according to claim 13, wherein the microorganism is a bacterium belonging to the genus Escherichia. ### Claim 17 (depends on 16) The method according to claim 16, wherein the microorganism is Escherichia coli. ### Claim 18 (depends on 1) The method according to claim 1, wherein the metabolites (X) are selected form the group consisting of vanillin, vanillic acid, melatonin, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, and creatine. ### Claim 19 (depends on 1) 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. ### Claim 20 (depends on 19) The method according to claim 19, 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. ### Claim 21 (depends on 1) 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. ### Claim 22 (depends on 1) 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. ### Claim 23 (depends on 22) The method according to claim 22, 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. ### Claim 24 (depends on 1) 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. ### Claim 25 (depends on 24) The method according to claim 24, wherein the L-cysteine biosynthesis enzyme 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. ### Claim 26 (depends on 24) The method according to claim 24, wherein the activity of the L-cysteine biosynthesis enzyme is increased by increasing the activity of a protein encoded by cysR gene. ### Claim 27 (depends on 1) The method according to claim 1, wherein the microorganism has been further modified so that the activity of a protein encoded by NCgl2048 gene is reduced as compared with a non-modified microorganism. ### Claim 28 (depends on 1) 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. ### Claim 29 (depends on 28) The method according to claim 28, wherein the microorganism is a bacterium belonging to the genus Corynebacterium. ### Claim 30 (depends on 28) The method according to claim 28, wherein the microorganism is Corynebacterium glutamicum.