Method for producing target substance

Patent metadata

Field	Value
Publication number	US-10155952-B2
Application number	US-201615134674-A
Country	US
Kind code	B2
Filing date	Apr 21, 2016
Priority date	Oct 23, 2013
Publication date	Dec 18, 2018
Grant date	Dec 18, 2018

Abstract

A method for producing a target substance is provided. A target substance is produced by culturing a coryneform bacterium, which is able to produce a target substance, and which also has an improved ability to assimilate xylose as a result of the introduction of a mutation into a coding region and/or an expression control region of the NCgl2954 gene on the chromosome of the bacterium. The culture is conducted in a medium containing xylose, and the target substance can be collected from the medium.

First claim

The invention claimed is:

1. A method for producing a target substance comprising:
culturing a coryneform bacterium having an ability to produce a target substance in a medium containing xylose to produce and accumulate the target substance in the medium; and
collecting the target substance from the medium,

wherein the bacterium has been modified so that the ability of the bacterium to assimilate xylose is improved as compared with a non-modified bacterium by attenuation of expression or disruption of the NCgl2954 gene on the chromosome of the bacterium.

2. The method according to claim 1 , wherein the ability of the bacterium to assimilate xylose has been improved by improving the ability of the bacterium to take up xylose.

3. The method according to claim 1 , wherein the NCgl2954 gene is a DNA encoding a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 14;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 14, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein when said protein is deleted from the coryneform bacterium, the bacterium has an improved ability to assimilate xylose; 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: 14, and wherein when said protein is deleted from the coryneform bacterium, the bacterium has an improved ability to assimilate xylose.

4. The method according to claim 1 , wherein said attenuation of expression or disruption is attained by introduction of a mutation into a coding region and/or an expression control region of the NCgl2954 gene that results in a mutation of the encoded amino acid sequence, and wherein the mutation of the encoded amino acid sequence is selected from the group consisting of:
(1) replacement of an amino acid residue corresponding to the proline leucine residue at position 438 of SEQ ID NO: 14 with an amino acid residue other than a leucine residue;
(2) replacement of an amino acid residue corresponding to the tryptophan residue at position 274 of SEQ ID NO: 14 with an amino acid residue other than a tryptophan residue;
(3) replacement of an amino acid residue corresponding to the tyrosine residue at position 377 of SEQ ID NO: 14 with an amino acid residue other than a tyrosine residue;
(4) replacement of an amino acid residue corresponding to the leucine residue at position 365 of SEQ ID NO: 14 with an amino acid residue other than a leucine residue;
(5) replacement of an amino acid residue corresponding to the leucine residue at position 366 of SEQ ID NO: 14 with an amino acid residue other than a leucine residue;
(6) replacement of an amino acid residue corresponding to the alanine residue at position 367 of SEQ ID NO: 14 with an amino acid residue other than an alanine residue;
(7) truncation of the N-terminus amino acid residues beginning with the amino acid residue at position 368 of SEQ ID NO: 14; and
(8) combinations thereof.

5. The method according to claim 4 , wherein:
said amino acid residue other than a leucine residue at position 438 of SEQ ID NO: 14 is proline;
said amino acid residue other than a tryptophan residue is arginine;
said amino acid residue other than a tyrosine residue is asparagine;
said amino acid residue other than a leucine residue at position 365 of SEQ ID NO: 14 is serine;
said amino acid residue other than a leucine residue at position 366 of SEQ ID NO: 14 is arginine; and
said amino acid residue other than an alanine residue is phenylalanine.

6. The method according to claim 1 , wherein the bacterium has been further modified so that activities or activity of xylose isomerase and/or xylulokinase is increased.

7. The method according to claim 6 , wherein the xylose isomerase is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 11;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 11, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein said protein has xylose isomerase 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: 11, and wherein said protein has xylose isomerase activity.

8. The method according to claim 6 , wherein the xylulokinase is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 12;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 12, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein said protein has xylulokinase 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: 12, and wherein said protein has xylulokinase activity.

9. The method according to claim 1 , wherein the bacterium has been further modified so that activity or activities of an enzyme selected from the group consisting of xylose dehydrogenase, xylonolactonase, xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, α-ketoglutaric semialdehyde dehydrogenase, and combinations thereof is/are increased.

10. The method according to claim 9 , wherein the xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutaric semialdehyde dehydrogenase are derived from an Escherichia bacterium, Sphingomonas bacterium, and Bacillus bacterium, respectively.

11. The method according to claim 9 , wherein the xylose dehydrogenase is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 16 or 42;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 16 or 42, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein said protein has xylose dehydrogenase 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: 16 or 42, and wherein said protein has xylose dehydrogenase activity.

12. The method according to claim 9 , wherein the xylonolactonase is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 18 or 44;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 18 or 44, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein said protein has xylonolactonase 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: 18 or 44, and wherein said protein has xylonolactonase activity.

13. The method according to claim 9 , wherein the xylonate dehydratase is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 20 or 46;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 20 or 46, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and wherein said protein has xylonate dehydratase 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: 20 or 46, and wherein said protein has xylonate dehydratase activity.

14. The method according to claim 9 , wherein the 2-keto-3-deoxyxylonate dehydratase is a protein selected from the group consisting of:
(A) a protein compr

Assignees

• Ajinomoto Kk

Inventors

• Yamada Kazuteru
• Hara Yoshihiko

Classifications

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/77 Primary

  for Corynebacterium; for Brevibacterium · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12P19/40

  having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides · CPC title