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Method for increasing yield of vitamin K2 by utilizing recombinant bacillus natto

A technology of Bacillus natto and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of insufficient metabolic flux, affecting the synthesis of vitamin K, complex synthesis pathways, etc.

Pending Publication Date: 2020-08-18
西宝生物科技(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, vitamin K 2 The synthetic pathway of vitamin K is very complex, and Bacillus natto synthesizes vitamin K 2 Insufficient metabolic flux will seriously affect vitamin K 2 Synthesis

Method used

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  • Method for increasing yield of vitamin K2 by utilizing recombinant bacillus natto
  • Method for increasing yield of vitamin K2 by utilizing recombinant bacillus natto
  • Method for increasing yield of vitamin K2 by utilizing recombinant bacillus natto

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction of recombinant bacteria NA1

[0039] The flutaroxine synthase gene mqnA (mqnA, genebank ID: 1099946) was overexpressed on the chromosome of Bacillus natto through the constitutive promoter P43. Using a marker-free genetic modification strategy, see the article (Yan, X., Yu, H.-J., Hong, Q., Li, S.P., 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microb. 74, 5556-5562), the specific construction process is as follows:

[0040] (1) Gene cloning

[0041] ① Using the Bacillus natto genome as a template, the upstream homology arm sequence mqnA-up of the mqnA gene was amplified using primers mqnA-up.FOR and mqnA-up.REV.

[0042] ② Artificially synthesize the lox71-zeo-lox66 cassette containing the bleomycin gene (sequence shown in SEQ ID NO.3).

[0043] ③ Using the Bacillus natto genome as a template, the P43 promoter sequence (sequence shown in SEQ ID NO.1) was amplified by using primers P43.For and P43...

Embodiment 2

[0054] Embodiment 2: the construction of recombinant bacterium NA2

[0055] On the basis of the bacterial strain NA1 that embodiment 1 obtains, adopt the method similar to embodiment 1, utilize P 43 Promoter overexpression of deoxyxanthine flutaloxine synthetase on Bacillus natto chromosome ( wxya , genebank ID: 1099767), the specific construction process is:

[0056] (1) Acquisition of fusion fragments

[0057] Using the Bacillus natto genome as a template, amplified separately to obtain wxya Gene upstream homology arm sequence mqnB-up , wxya gene fragment and P 43 Promoter sequence, artificially synthesized lox71-zeo-lox66 box sequence (sequence shown in SEQ ID NO.2), and then four fragments mqnB-up, lox71-zeo-lox66 box, P 43 promoter sequence, wxya Gene fragments were subjected to overlap extension PCR to obtain fusion gene fragments wxya up -lox71-zeo-lox66-P 43 - mqnB-mqnB down .

[0058] (2) Homologous recombination

[0059] Transform the fusion fragment ...

Embodiment 3

[0060] Embodiment 3: the construction of recombinant bacterium NA3

[0061] On the basis of the bacterial strain NA2 that embodiment 2 obtains, adopt the similar method with embodiment 1, utilize P hbs The promoter (sequence shown in SEQ ID NO.5) replaces the O-succinylbenzoic acid-CoA ligase in Bacillus natto ( QUR , genebankID: 1099990) the natural promoter of the gene, the specific construction process is as follows:

[0062] (1) Acquisition of fusion fragments

[0063] Using the Bacillus natto genome as a template, amplified separately to obtain QUR Gene upstream homology arm sequence mqnC-up , QUR Gene fragments and P hbs Promoter sequence, artificially synthesized lox71-zeo-lox66 box sequence (sequence shown in SEQ ID NO.2). Then the four fragments mqnC-up, lox71-zeo-lox66 cassette, P hbs promoter sequence, QUR Gene fragments were subjected to overlap extension PCR to obtain fusion gene fragments QUR up -lox71-zeo-lox66- P hbs - QUR .

[0064] (2)...

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Abstract

The invention discloses a method for increasing the yield of vitamin K2 by utilizing recombinant bacillus natto. The method comprises the following steps: overexpressing a flutalosin synthetase gene mqnA on a bacillus natto chromosome through a constitutive promoter P43; on the basis of the obtained strain NA1, the strain NA1 is obtained; the method comprises the following steps: overexpressing agene of deoxyxanthine flutalosin synthetase on a bacillus natto chromosome by using a P43 promoter; on the basis of the obtained bacterial strain NA2; a Phbs promoter is used for replacing a natural promoter of an O-succinylbenzoic acid-CoA ligase gene in bacillus natto; on the basis of the obtained strain NA3, isobranching acid genes on bacillus natto chromosomes are replaced with 1, 4-dihydroxy-6-naphthoic acid synthase genes which contain Phbs promoters and are derived from streptomyces coelicolor, and a seed solution of the recombinant bacteria is inoculated into a fermentation culture medium according to the inoculation amount of 3%-6%. According to the invention, four recombinant bacteria NA1-NA4 are constructed, wherein the NA3-NA4 significantly improves the yield of vitamin K2.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for improving vitamin K by using recombinant Bacillus natto 2 yield method. Background technique [0002] Vitamin K 2 , is one of the active forms of vitamin K. It is an important class of fat-soluble vitamins connected by a menaquinone parent ring and n isoprene side chains. The cofactor has the characteristics of long half-life and good bioavailability. Anti-arterial calcification and osteoarthritis, anti-tumor, anti-oxidation, anti-aging, skin beauty, treatment of hepatitis, adjustment of digestive tract, relief of smooth muscle spasm, treatment and prevention of osteoporosis, prevention of liver cirrhosis from progressing to liver cancer, vitamin treatment K 2 It plays an important role in deficient hemorrhage, diuresis, liver detoxification, and lowering blood pressure. Because menadione has better affinity and longer half-life to the human body, it...

Claims

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Application Information

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IPC IPC(8): C12N15/67C12N15/75C12P7/66C12R1/07
CPCC12N15/67C12N15/75C12N9/93C12Y602/01026C12N9/00C12P7/66
Inventor 阮月敏区文彩陆阳其他发明人请求不公开姓名
Owner 西宝生物科技(上海)股份有限公司
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