Useful microorganism and method for producing substance of interest

Patent metadata

Field	Value
Publication number	US-10047382-B2
Application number	US-201314408400-A
Country	US
Kind code	B2
Filing date	Jul 3, 2013
Priority date	Jul 3, 2012
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

Abstract

It is an object of the present invention to provide a bacterial strain that can decrease the amount of an intermediate Compound P converted into Metabolite M and efficiently accumulate Compound P in a medium that is not supplemented with Metabolite M or the final product generated from Metabolite M. The present invention provides a prokaryotic organism having all features (a) to (d) as defined in the specification so as to accumulate Compound P by regulating expression level of Enzyme X that converts Compound P as an intermediate metabolite into Metabolite M in a biosynthetic pathway in which Metabolite M indispensable for the growth is produced from a carbon source.

First claim

The invention claimed is:

1. A genetically-modified prokaryotic organism comprising features (a) to (d) below:
(a) activity of at least one enzyme necessary for the production of 3-dehydroshikimate selected from 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, phosphoenolpyruvic acid synthetase, phosphoenolpyruvic acid carboxykinase and transketolase is enhanced compared with that of a corresponding wild-type organism;
(b) activity of a first shikimate dehydrogenase is lost or reduced by mutation to a translation region or promoter of the gene encoding the first shikimate dehydrogenase;
(c) a vanA, rhcH, or peal gene promoter is functionally linked to a gene encoding a second shikimate dehydrogenase, and
(d) a benA gene promoter or a nagI gene promoter is functionally linked to a gene encoding vanR, rhcR, or pcaR.

2. The prokaryotic organism according to claim 1 , wherein activity of one or more metabolic enzymes that uses shikimic acid, chorismic acid, prephenic acid, anthranilic acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, or arogenic acid as a substrate is lost or reduced.

3. The prokaryotic organism according to claim 1 , wherein the prokaryotic organism expresses vanR, rhcR or pcaR and the activity of vanR, rhcR or pcaR is repressed by exposure to ferulic acid, vanillic acid, vanillin, benzoic acid, 3-hydroxybenzoic acid, resorcinol, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, fructose, and/or sucrose.

4. The prokaryotic organism according to claim 1 , wherein the gene encoding vanR, rhcR, or pcaR is from the Corynebacterium glutamicum ATCC13032 strain.

5. The prokaryotic organism according to claim 1 , wherein the vanA, rhcH or peal gene promoter functionally linked to a gene encoding a second shikimate dehydrogenase is from the Corynebacterium glutamicum ATCC13032 strain.

6. The prokaryotic organism according to claim 1 having the feature (e) below in addition to the features (a) to (d):
(e) a benA gene promoter or a nagI gene promoter is functionally linked to the qsuB gene encoding dehydroshikimate dehydratase.

7. The prokaryotic organism according to claim 6 , wherein activity of one or more metabolic enzymes among the enzymes of the prokaryotic organism that metabolize protocatechuic acid is lost or reduced.

8. The prokaryotic organism according to claim 6 , wherein the genetically-modified prokaryotic organism expresses the qsuB gene and the expression is induced by exposure to ferulic acid, vanillic acid, vanillin, benzoic acid, 3-hydroxybenzoic acid, resorcinol, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, fructose, and/or sucrose.

9. The prokaryotic organism according to claim 1 , which is Corynebacterium glutamicum or Escherichia coli.

10. The prokaryotic organism according to claim 1 , wherein for feature (b) the first shikimate dehydrogenase is quinate/shikimate dehydrogenase and the mutation is to the translation region or the promoter of the qsuD gene.

11. The prokaryotic organism according to claim 1 , further comprising the feature in which the activity of dehydroshikimate dehydratase is lost or reduced by mutation to a translation region or promoter of the qsuB gene encoding dehydroshikimate dehydratase.

12. The genetically-modified prokaryotic organism according to claim 11 , wherein the first shikimate dehydrogenase is quinate/shikimate dehydrogenase and the mutation is to the translation region or the promoter of the qsuD gene.

13. The prokaryotic organism according to claim 12 that is able to grow in media not supplemented with phenylalanine, tyrosine, and tryptophan above concentrations required for the growth of a corresponding wild-type organism.

14. The prokaryotic organism according to claim 1 that can produce 11.2 g/L or more of protocatechuic acid.

15. The prokaryotic organism according to claim 12 that can produce 15.4 g/L of 3-dehydroshikimate.

16. A method for producing 3-dehydroshikimic acid and/or protocatechuic acid comprising culturing the genetically-modified prokaryotic organism according to claim 1 , wherein the genetically-modified prokaryotic organism expresses vanR, rhcR, or pcaR.

17. A method for producing 3-dehydroshikimic acid and/or protocatechuic acid comprising culturing the genetically-modified prokaryotic organism according to claim 1 , wherein the genetically-modified prokaryotic organism expresses vanR, rhcR, or pcaR in response to activation of the benA gene promoter or nagI gene promoter by exposure to 3-hydroxybenzoic acid or benzoic acid.

18. A method for producing 3-dehydroshikimic acid and/or protocatechuic acid comprising culturing the genetically-modified prokaryotic organism according to claim 1 , wherein the genetically-modified prokaryotic organism expresses vanR, rhcR, or pcaR in response to activation of the benA gene promoter or nagI gene promoter but the second shikimate dehydrogenase is still expressed by one of the following:
reducing the culture temperature to a level at which vanR, rhcR, or pcaR are inhibited, or adding ferulic acid, vanillic acid, vanillin, resorcinol, 2,4-dihydroxybenzoic acid, or 4-dihydroxybenzoic acid in amounts that inhibit vanR, rhcR, or pcaR.

Assignees

• Kao Corp

Inventors

• IIDA Kougo
• Iwasaki Takumi
• Nishi Tatsunari

Classifications

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title

• C12P13/14

  Glutamic acid; Glutamine · CPC title

• C12P13/20

  Aspartic acid; Asparagine · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P13/06

  Alanine; Leucine; Isoleucine; Serine; Homoserine · CPC title