Method for producing lower alkyl ester

Patent metadata

Field	Value
Publication number	US-9708637-B2
Application number	US-201615164381-A
Country	US
Kind code	B2
Filing date	May 25, 2016
Priority date	Sep 27, 2007
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

A method for producing an L-amino acid is described, for example, L-phenylalanine and L-histidine, by fermentation using a bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified by attaching a DNA fragment able to be transcribed encoding the peptide represented in SEQ ID NO: 2, or a variant thereof, particularly a portion of the ssrA gene, to the 3′-end of gene encoding for the bacterial enzyme, which influences on the L-amino acid biosynthesis, such as chorismate mutase/prephenate dehydrogenase or phosphoglucose isomerase.

First claim

We claim:

1. A method for producing a lower alkyl ester of α-L-aspartyl-L-phenylalanine, comprising:
A) cultivating a bacterium of Escherichia coli in a culture medium which is able to produce and accumulate L-phenylalanine in the medium, and
B) synthesizing the lower alkyl ester of α-L-aspartyl-L-phenylalanine from aspartic acid or a derivative thereof and the L-phenylalanine obtained in step A);
wherein the bacterium comprises a DNA comprising:
i) a gene selected from the group consisting of tyrA, pheA, pgi, ilvE, ilvA, tdcB, sdaA, sdaB, argA, argG, proB, thrB, and combinations thereof, and
ii) a DNA fragment able to be transcribed and encoding the peptide of SEQ ID NO: 2, or a variant thereof consisting of a deletion, insertion, substitution, or addition of 1 amino acid as compared to SEQ ID NO: 2; and
wherein said DNA fragment of ii) is attached to the 3′ end of said gene of i) and consequently enhances production of an L-amino acid.

2. The method according to claim 1 , wherein said bacterium is an L-phenylalanine producing bacterium.

3. The method according to claim 1 , wherein said gene is tyrA, which encodes the bifunctional enzyme chorismate mutase/prephenate dehydrogenase.

4. The method according to claim 1 , wherein said bacterium is an L-histidine-producing bacterium.

5. The method according to claim 1 , wherein said gene is pgi which encodes a phosphoglucose isomerase enzyme.

6. The method according to claim 1 , wherein the variant is the peptide of SEQ ID NO: 15.

7. The method according to claim 1 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

8. The method according to claim 2 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

9. The method according to claim 3 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

10. The method according to claim 4 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

11. The method according to claim 5 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

12. The method according to claim 6 , wherein said synthesizing in step B) further comprises:
C) esterifying the L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
D) condensing the lower alkyl ester of L-phenylalanine with the aspartic acid derivative in a condensation reaction mixture, wherein the derivative is N-acyl-L-aspartic anhydride,
E) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the condensation reaction mixture, and
F) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

Assignees

• Ajinomoto Kk

Inventors

• Shakulov Rustem Saidovich
• Klyachko Elena Vitalievna
• Doroshenko Vera Georgievna
• Airikh Larisa Gotlibovna

Classifications

• C12Y103/01013

  Prephenate dehydrogenase (NADP+) (1.3.1.13) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/001

  acting on the CH-CH group of donors (1.3) · CPC title

• C12Y503/01009

  Glucose-6-phosphate isomerase (5.3.1.9) · CPC title

• C12P13/222

  Phenylalanine · CPC title