Cofactor self-sufficient escherichia coli and construction method and application thereof

Patent metadata

Field	Value
Publication number	US-2023084966-A1
Application number	US-202217823853-A
Country	US
Kind code	A1
Filing date	Aug 31, 2022
Priority date	Sep 1, 2021
Publication date	Mar 16, 2023
Grant date	—

Abstract

A cofactor self-sufficient Escherichia coli and its construction method and application in the synthesis of L-glufosinate are provided. The present invention expresses a NADH kinase and key enzymes of the cofactor synthesis pathway in E. coli , and knocks out the genes of enzymes that catabolizes cofactor, and with the addition of co-metabolic intermediates during cell incubation, the intracellular NADP(H) concentration is increased by at least 50% and the catalytic activity of glutamate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the-glufosinate synthesis reaction.

First claim

What is claimed is:

1 . A cofactor self-sufficient Escherichia coli , obtained by constructing methods as follows,
(1) constructing expression vectors, transforming a NADH kinase gene, a glucose dehydrogenase gene, a glutamate dehydrogenase gene and a gene of NADP cofactor synthesis pathway into an Escherichia coli named E. coli BL21 (DE3), screening the correct transformants, and obtaining a glutamate dehydrogenase-glucose dehydrogenase-NADH kinase co-expressing gene engineering bacterium; said NADH kinase gene sequence is one of SEQ ID No. 1 to 5, said glucose dehydrogenase gene sequence is as shown in SEQ ID No. 6, said glutamate dehydrogenase gene sequence is as shown in SEQ ID No. 7, and said NADP cofactor synthesis pathway gene sequence is as shown in one of SEQ ID No. 8 to 10; (2) knocking out of any one or a combination of mazG, mudC, nadR genes in the genome of the co-expressing gene engineering bacterium to obtain said cofactor self-sufficient Escherichia coli.

2 . The cofactor self-sufficient Escherichia coli of claim 1 , wherein the sequence of said NADH kinase gene in step ( 1 ) is as shown in SEQ ID No. 3, and the sequence of said NADP cofactor synthesis pathway gene is as shown in SEQ ID No. 8.

3 . The cofactor self-sufficient Escherichia coli of claim 1 , wherein the mazG and nadR genes are knocked out in step ( 2 ).

4 . A method for constructing the cofactor self-sufficient Escherichia coli of claim 1 , comprising:
(1) constructing expression vectors, transforming a NADH kinase gene, a glucose dehydrogenase gene, a glutamate dehydrogenase gene and a gene of the NADP cofactor synthesis pathway into E. coli BL21 (DE3), screening the correct transformants, and obtaining a glutamate dehydrogenase-glucose dehydrogenase-NADH kinase co-expressing gene engineering bacterium; said NADH kinase gene sequence is as one of SEQ ID No. 1 to 5, said glucose dehydrogenase gene sequence is as shown in SEQ ID No. 6, said glutamate dehydrogenase gene sequence is as shown in SEQ ID NO. 7, and said NADP cofactor synthesis pathway gene sequence is as shown in one of SEQ ID No. 8 to 10; (2) knocking out of any one or a combination of mazG, mudC, nadR in the genome of the co-expressing gene-engineering bacterium to obtain said cofactor self-sufficient Escherichia coli.

5 . The method as claimed in claim 4 , wherein the sequence of said NADH kinase gene in step ( 1 ) is as shown in SEQ ID No. 3 and the gene sequence of said NADP cofactor synthesis pathway is as shown in SEQ ID No. 8; the mazG and nadR genes are knocked out in step ( 2 ).

6 . An application of the cofactor self-sufficient Escherichia coli of claim 1 in the preparation of L-glufosinate by microbial fermentation.

7 . The application of claim 6 , wherein said application is: using the wet bacterial cells obtained by fermentation culture of said cofactor self-sufficient Escherichia coli or the enzyme solution extracted by ultrasonic crushing of said wet bacterial cells as the catalyst, using 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid as the substrate, adding ammonium sulfate and glucose, using pH 7.5 buffer as the reaction medium to constitute the reaction system, and reacting at 35° C.˜40° C., 500˜600 rpm; after the reaction, the reaction solution is separated and purified to obtain L-glufosinate.

8 . The application of claim 7 , wherein in said reaction system, the amount of catalyst is 10˜50 g/L by total weight of wet bacterial cells, the initial concentration of substrate is 10˜500 mM, the amount of glucose addition is 12˜750 mM, and the amount of ammonium sulfate addition is 50 mM˜1.5M.

9 . The application of claim 7 , wherein in said reaction system, the amount of catalyst is 15 g/L by total weight of wet bacterial cells, the initial concentration of substrate is 200 mM, the amount of glucose addition is 250 mM, and the amount of ammonium sulfate addition is 300 mM.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Zou Shuping
• Jiang Zhentao
• Xu Jianmiao
• Cheng Feng
• Zheng Yuguo

Classifications

• C12N15/70 Primary

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

• C12Y101/01119 Primary

  Glucose 1-dehydrogenase (NADP+) (1.1.1.119) · CPC title

• C12Y207/01086

  NADH kinase (2.7.1.86) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y104/01002

  Glutamate dehydrogenase (1.4.1.2) · CPC title