Method for increasing yield of L-arginine by knocking out Flavin reductases

Patent metadata

Field	Value
Publication number	US-10465218-B2
Application number	US-201616063639-A
Country	US
Kind code	B2
Filing date	Jul 29, 2016
Priority date	Jul 26, 2016
Publication date	Nov 5, 2019
Grant date	Nov 5, 2019

Abstract

The invention discloses a method for increasing the yield of L-arginine by knocking out flavin reductases, and belongs to the technical field of amino acid production by microbial fermentation. Genes frd1 and frd2 for encoding hypothetic NADPH-dependent FMN reductase in Corynebacterium crenatum SDNN403 are over-expressed in E. coli BL21 and are purified to form target proteins Frd181 and Frd188, and functions of the target proteins are identified to obtain a result showing that the proteins Frd181 and Frd188 both are NAD(P)H-dependent flavin reductases producing H2O2. By taking a genome of the Corynebacterium crenatum SDNN403 as a template, frd1 and frd2 gene deletion fragments are obtained by overlap extension PCR; connecting pK18mobsacB to obtain knockout plasmids pK18mobsacB-Δfrd1 and pK18mobsacB-Δfrd2; carrying out electric shock to transform the Corynebacterium crenatum SDNN403; and carrying out secondary screening to obtain recombinant strains 403Δfrd1 and 403Δfrd2. Found by flask shaking fermentation, the yield of L-arginine is obviously increased by knocking out the genes frd1 and frd2.

First claim

What is claimed is:

1. A recombinant strain of Corynebacterium crenatum comprising a gene knockout of NADPH-dependent flavin mononucleotide (FMN) reductase gene frd1, or frd2, or both frd1 and frd2.

2. The recombinant strain of the Corynebacterium crenatum of claim 1 , wherein amino acid sequences of the NADPH-dependent FMN reductase genes frd1 and frd2 are respectively amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4.

3. The recombinant strain of the Corynebacterium crenatum of claim 1 , wherein the recombinant strain of the Corynebacterium crenatum is obtained by knocking out NADPH-dependent FMN reductase genes in Corynebacterium crenatum CGMCC NO:0890.

4. The recombinant strain of the Corynebacterium crenatum of claim 1 , wherein nucleotide sequences of the NADPH-dependent FMN reductase genes frd1 and frd2 are respectively nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:3.

5. A method for synthesizing L-arginine, comprising culturing a recombinant strain of Corynebacterium crenatum of claim 1 as a production strain.

6. The method of claim 5 , wherein:
the culturing step is performed at a temperature of 28° C. to 32° C.; and
the culturing step is performed in a medium comprising:
120 g/L glucose,
40 g/L corn steep liquor,
8×10 −5 g/L biotin,
5×10 −4 g/L histidine,
0.02 g/L manganese sulfate,
20 g/L ammonium sulfate,
0.5 g/L magnesium sulfate,
1.5 g/L monopotassium phosphate, and
0.02 g/L ferrous sulfate.

7. A method for promoting synthesis of L-arginine by knocking out flavin reductases, comprising:
knocking out NADPH-dependent FMN reductase genes frd1, or frd2, or both frd1 and frd2 in Corynebacterium crenatum to obtain a recombinant strain, and
synthesizing L-arginine by culturing the recombinant strain as a production strain.

8. The method of claim 7 , wherein amino acid sequences of the NADPH-dependent FMN reductase genes frd1 and frd2 are respectively amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4.

9. The method of claim 7 , wherein the Corynebacterium crenatum is Corynebacterium crenatum CGMCC NO:0890.

10. A method of use of the recombinant strain of the Corynebacterium crenatum of claim 1 to medicines, food or feed industry, comprising culturing the recombinant strain of Corynebacterium crenatum, and synthesizing L-arginine from a culture thereof.

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Man Zaiwei
• Xu Meijuan
• Yang Taowei
• Zhang Xian

Classifications

• C12Y105/01038

  FMN reductase (NADPH) (1.5.1.38) · CPC title

• C12N9/0028 Primary

  with NAD or NADP as acceptor (1.5.1) · CPC title

• C12P13/10 Primary

  Citrulline; Arginine; Ornithine · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title