Machine learning gene mining method and phosphinothricin dehydrogenase mutant for amino translocation

Patent metadata

Field	Value
Publication number	US-11781117-B2
Application number	US-202117505945-A
Country	US
Kind code	B2
Filing date	Oct 20, 2021
Priority date	Dec 31, 2020
Publication date	Oct 10, 2023
Grant date	Oct 10, 2023

Abstract

Disclosed are a machine learning gene mining method and a phosphinothricin dehydrogenase mutant for amino translocation. The phosphinothricin dehydrogenase mutant for amino translocation is obtained by mutation of a wild-type phosphinothricin dehydrogenase with an amino acid sequence as shown in SEQ ID No.2 at one of the following sites: (1) E263D-K134R-H96A-R290V; (2) E263D-K134R-H96A; (3) E263D-K134R; (4) E263D; (5) E263N; (6) E263C; and (7) E263G. The present invention utilizes the site-saturation mutagenesis technology to mutate a phosphinothricin dehydrogenase gene as shown in SEQ ID No. 1, finds that the 263rd, 134th, 290th and 290th positions are the key sites affecting enzyme activity and stereoselectivity, and obtains a mutant with enzyme activity and ee value much higher than those of the parent phosphinothricin dehydrogenase.

First claim

What is claimed is:

1. A phosphinothricin dehydrogenase mutant for amino translocation with the amino acid sequence of wild-type phosphinothricin dehydrogenase from Pseudomonas hunanensis as shown in SEQ ID NO: 2 except having one of the following sites of mutation:
(1) E263D-K134R-H96A-R290V;
(2) E263D-K134R-H96A;
(3) E263D-K134R;
(4) E263D;
(5) E263N;
(6) E263C; or
(7) E263G.

2. A gene encoding the phosphinothricin dehydrogenase mutant for amino translocation according to claim 1 .

3. A genetically recombinant bacterium comprising a host cell and a target gene transformed into the host cell, wherein the target gene comprises the gene according to claim 2 .

4. The genetically recombinant bacterium according to claim 3 , wherein the genetically recombinant bacterium further comprises a gene encoding a glucose dehydrogenase transformed into the host cell.

5. The phosphinothricin dehydrogenase mutant for amino translocation according to claim 1 , wherein the site of mutation is: E263D-K134R-H96A-R290V.

6. A method for preparing L-phosphinothricin, by reacting 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate as catalyzed by a catalyst in the presence of an inorganic amino donor, a coenzyme circulation system and a corresponding co-substrate of the coenzyme circulation system to obtain L-phosphinothricin; wherein
the catalyst is one of:
(1) the phosphinothricin dehydrogenase mutant for amino translocation according to claim 1 ; and
(2) a genetically recombinant bacterium producing the phosphinothricin dehydrogenase mutant for amino translocation according to claim 1 , or a crude enzyme liquid containing the phosphinothricin dehydrogenase mutant obtained by lysis of the genetically recombinant bacterium.

7. The method according to claim 6 , wherein the coenzyme circulation system is at least one of:
(1) a formate dehydrogenase coenzyme circulation system comprising a formate dehydrogenase, formate and a coenzyme;
(2) a glucose dehydrogenase coenzyme circulation system comprising a glucose dehydrogenase, glucose and a coenzyme; and
(3) an alcohol dehydrogenase coenzyme circulation system comprising an alcohol dehydrogenase, isopropanol and a coenzyme.

8. The method according to claim 7 , wherein the coenzyme circulation system is a formate dehydrogenase coenzyme circulation system comprising a formate dehydrogenase, formate and a coenzyme.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Cheng Feng
• Wu Dongyang
• Zou Shuping
• Xu Jianmiao
• Zheng Yuguo

Classifications

• C12N9/0016 Primary

  with NAD or NADP as acceptor (1.4.1) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• G16B30/10

  Sequence alignment; Homology search · CPC title

• C12Y104/01002

  Glutamate dehydrogenase (1.4.1.2) · CPC title