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

Patent metadata

Field	Value
Publication number	US-12104191-B2
Application number	US-202217823853-A
Country	US
Kind code	B2
Filing date	Aug 31, 2022
Priority date	Sep 1, 2021
Publication date	Oct 1, 2024
Grant date	Oct 1, 2024

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 glufosinate 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 a constructing methods as follows:
(1) constructing one or more plasmids or expression vectors capable of intracellular linearization and self-recombination that comprise target genes including a NADH kinase gene, a glucose dehydrogenase gene, a glufosinate dehydrogenase gene and a gene of a NADP cofactor synthesis pathway;
transforming the one or more plasmids or expression vectors comprising the target genes into an Escherichia coli named E. coli BL21 (DE3); and
obtaining a transformed Escherichia coli with introduction of the one or more plasmids or expression vectors comprising the target genes that is a glufosinate dehydrogenase-glucose dehydrogenase-NADH kinase co-expressing gene engineering bacterium; wherein said NADH kinase gene sequence is one of SEQ ID NOS: 1 to 3, said glucose dehydrogenase gene sequence is as shown in SEQ ID NO: 6, said glufosinate 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 NOS: 8 and 10; and
(2) knocking out of any one or a combination of mazG and 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 preparing L-glufosinate, comprising:
performing a fermentation culture of the cofactor self-sufficient Escherichia coli of claim 1 , and preparing wet bacterial cells of the cofactor self-sufficient Escherichia coli from the fermentation culture;
preparing a reaction medium containing the wet bacterial cells or an enzyme solution obtained by ultrasonic crushing of the wet bacterial cells as a catalyst, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid as a substrate, ammonium sulfate, and glucose, wherein the reaction medium is buffered at pH 7.5;
reacting the reaction medium at 35 to 40° C. with shaking at 500 to 600 rpm; and
purifying L-glufosinate from the reaction medium.

5. The method of claim 4 , wherein the reaction medium comprises the wet bacterial cells at a total weight of 10 to 50 g/L, an initial concentration of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid from 10 to 500 mM, glucose at a concentration of 12 to 750 mM, and ammonium sulfate at a concentration of 50 mM to 1.5 M.

6. The method of claim 4 , wherein the reaction medium comprises the wet bacterial cells at a total weight of 15 g/L, an initial concentration of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid is 200 mM, glucose at a concentration of 250 mM, and ammonium sulfate at a concentration of 300 mM.

7. A method for constructing a cofactor self-sufficient Escherichia coli , comprising:
(1) constructing one or more plasmids or expression vectors capable of intracellular linearization and self-recombination that comprise target genes including a NADH kinase gene, a glucose dehydrogenase gene, a glufosinate dehydrogenase gene and a gene of a NADP cofactor synthesis pathway, transforming the one or more plasmids or expression vectors comprising the target genes into Escherichia coli BL21 (DE3), and obtaining a transformed Escherichia coli with introduction of the one or more plasmids or expression vectors comprising the target genes that is a glufosinate dehydrogenase-glucose dehydrogenase-NADH kinase co-expressing gene engineering bacterium; wherein said NADH kinase gene sequence is as one of SEQ ID NOS: 1 to 5, said glucose dehydrogenase gene sequence is as shown in SEQ ID NO: 6, said glufosinate dehydrogenase gene sequence is as shown in SEQ ID NO: NO. 7, and said NADP cofactor synthesis pathway gene sequence is as shown in one of SEQ ID NO: 8 to 10; and
(2) knocking out of any one or a combination of mazG, and nadR in the genome of the co-expressing gene-engineering bacterium to obtain said cofactor self-sufficient Escherichia coli.

8. The method as claimed in claim 7 , wherein the sequence of said NADH kinase gene in step (1) is as shown in SEQ ID NO: 3 the gene sequence of said gene of NADP cofactor synthesis pathway is as shown in SEQ ID NO: 8; and the mazG and nadR genes are knocked out in step (2).

Assignees

• Univ Zhejiang Technology

Inventors

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

Classifications

• C12Y207/07018

  Nicotinate-nucleotide adenylyltransferase (2.7.7.18) · CPC title

• C12Y204/02011

  Nicotinate phosphoribosyltransferase (2.4.2.11) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N9/1077

  Pentosyltransferases (2.4.2) · CPC title