Method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multi-enzyme coupling

Patent metadata

Field	Value
Publication number	US-12215372-B2
Application number	US-202017607371-A
Country	US
Kind code	B2
Filing date	Jun 8, 2020
Priority date	Mar 18, 2020
Publication date	Feb 4, 2025
Grant date	Feb 4, 2025

Abstract

A method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multienzyme coupling, where D,L-phosphinothricin as a raw material is catalyzed by an enzyme catalysis system to obtain L-phosphinothricin, wherein the enzyme catalysis system comprises a D-amino acid oxidase mutant for catalyzing D-phosphinothricin in D,L-phosphinothricin into 2-carbonyl-4-[hydroxy(methyl)phosphono] butyric acid and a transaminase for catalytic reduction of the 2-carbonyl-4-[hydroxy(methyl)phosphono] butyric acid into L-phosphinothricin; the D-amino acid oxidase mutant is obtained by mutation of D-amino acid oxidase in wild strain Rhodotorula taiwanensis at one of the following sites: (1) M213S-N54V-F58E; (2) M213S-N54V-F58E-D207A; (3) M213S-N54V-F58E-D207A-S60T. According to the present invention, the D-amino acid oxidase mutant provides better catalytic efficiency, and when racemic D,L-phosphinothricin is used as a substrate for catalytic reaction, the conversion rate is much higher than that of the wild type enzyme, and the PPO yield is also greatly improved.

First claim

What is claimed is:

1. A method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multi-enzyme coupling, where D,L-phosphinothricin as a raw material is catalyzed by an enzyme catalysis system to obtain L-phosphinothricin, comprising the step of catalyzing D-phosphinothricin in D,L-phosphinothricin into 2-carbonyL-4-[hydroxy(methyl)phosphono] butyric acid by a D-amino acid oxidase mutant of the enzyme catalysis system and catalytically reducing the 2-carbonyl-4-[hydroxy(methyl)phosphono] butyric acid into L-phosphinothricin by a transaminase of the enzyme catalysis system, and
wherein the D-amino acid oxidase mutant is obtained by mutation of D-amino acid oxidase in wild strain Rhodotorula taiwanensis at one of the following sites:
(a) M213S-N54V-F58E-D207A;
(b) M213S-N54V-F58E-D207A-S60T.

2. The method according to claim 1 , wherein the transaminase has the amino acid sequence as shown in SEQ ID NO: 39.

3. The method according to claim 2 , wherein the D-amino acid oxidase mutant is obtained by adding a genetically engineered bacterium expressing the D-amino acid oxidase mutant into a reaction system; and
the transaminase is obtained by adding a genetically engineered bacterium expressing the transaminase together with a coenzyme pyridoxal phosphate into the reaction system.

4. The method according to claim 3 , wherein E. coli BL21 (DE3) is used as a host cell for both of the genetically engineered bacteria.

5. The method according to claim 3 , wherein in the reaction system, the D,L-phosphinothricin has a final concentration of 100-400 mM, the genetically engineered bacterium expressing the D-amino acid oxidase mutant is added at an amount of 20-40 g/L, the genetically engineered bacterium expressing the transaminase is added at an amount of 30-50 g/L, and the coenzyme pyridoxal phosphate has a concentration of 1 mM.

6. The method according to claim 5 , wherein the reaction is carried out at a temperature of 30° C. and a pH of 8 for 10 hours.

7. The method according to claim 6 , wherein after the reaction is completed, the final product L-phosphinothricin is separated and extracted by ion exchange and crystallization.

8. A D-amino acid oxidase mutant obtained by mutation of a D-amino acid oxidase in wild-type Rhodotorula taiwanensis at one of the following sites:
(a) M213S-N54V-F58E-D207A;
(b) M213S-N54V-F58E-D207A-S60T.

9. A gene encoding the D-amino acid oxidase mutant according to claim 8 , wherein the gene has the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

10. A genetically engineered bacterium comprising the gene according to claim 9 .

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Cheng Feng
• Wang Liuyu
• Zheng Yuguo
• Xu Jianmiao
• Zou Shuping

Classifications

• C12Y206/01019

  4-Aminobutyrate—2-oxoglutarate transaminase (2.6.1.19) · CPC title

• C12Y104/03003 Primary

  D-Amino-acid oxidase (1.4.3.3) · CPC title

• C12N2800/101

  for bacteria · CPC title

• C12N2511/00

  Cells for large scale production · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title