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Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain

A Bacillus subtilis and genetic modification technology, applied in the field of genetic modification of Bacillus subtilis, can solve the problems of inability to accurately modify target genes, long period of genetic breeding and high probability of secondary mutation

Pending Publication Date: 2020-07-31
TIANJIN UNIV MARINE TECH RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, traditional mutagenesis or fermentation optimization of Bacillus natto is basically used to produce MK-7. These methods can increase the yield of MK-7, but they cannot precisely modify the target gene, and the probability of secondary mutation is high , the cycle of genetic breeding is longer

Method used

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  • Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain
  • Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain
  • Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain

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Experimental program
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Embodiment Construction

[0012] (1) Materials

[0013] Strains, Plasmids and Media

[0014] See Table 1 for details of all bacterial strains and plasmid information involved in this example.

[0015] LB medium (peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L) is used for general cultivation of B. subtilis, solid medium is added with 15g / L agar powder, neomycin 16μg / mL when needed , or chloramphenicol 8 μg / mL. Fermentation medium: glycerol 30mL / L, soybean peptone 60g / L, yeast extract 5g / L, K 2 HPO 4 3g / L, MgSO 4 ·7H 2 O0.5g / L, pH7.3.

[0016] The strains and plasmids involved in the experiments in Table 1

[0017]

[0018]

[0019] Reagents and instruments

[0020] FastTaq enzyme, Hifi DNA polymerase, and dNTP were all purchased from Beijing Quanshijin Biotechnology Co., Ltd.; bacterial total RNA extraction kit TRIzol Reagent was purchased from Invitrogen; ReverTra Ace qPCR RT Kit and SYBRGreen Real-time PCR Master Mix kit were purchased from From TOYOBO Company; standard MK-7 ...

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Abstract

The invention discloses a method for genetically modifying bacillus subtilis. The method comprises the following steps: (1) constructing an original strain MK3-MEP123-delta dhbB; (2) carrying out overexpression on a glycerol kinase gene glpK and a glycerol-3-phosphate dehydrogenase gene glpD; and (3) knocking out the encoding gene mgsA of the pyruvic aldehyde synthetase and the encoding gene araMof glycerol-1-phosphate dehydrogenase. According to the method, glpK and glpD are overexpressed in sequence, the utilization rate of glycerol is increased, synthesis of dihydroxyacetone phosphoric acid is promoted, and then synthesis of MK-7 is increased; mgsA and areM are knocked out in sequence, synthesis of pyruvic aldehyde and synthesis of glycerol 1-phosphoric acid are blocked, so that consumption of dihydroxyacetone phosphoric acid is reduced, and synthesis of MK-7 is promoted. After the obtained recombinant strain BSMK_4 is fermented in a 500 mL shake flask for 96 hours, the yield of the MK-7 is 70.3 mg / L and is increased by 26.4% compared with that of a starting strain MK3-MEP123-delta dhbB (55.6 mg / L).

Description

Technical field: [0001] The invention relates to a method for genetically transforming bacillus subtilis, belonging to the field of biological genetic engineering. Background technique: [0002] Natural fat-soluble vitamin K including plant sources of vitamin K 1 (also known as phylloquinone, PK) and vitamin K from bacterial sources 2 (Also known as Menadione, MK). According to the number of isoprene units in the side chain, there are 14 kinds of menadione, which are recorded as MK-n, and the common ones are MK-4 and MK-7. In prokaryotes, MK-n participates in the electron transport of the respiratory chain. For humans and other mammals, since vitamin K is an important cofactor for the translation of glutamic acid residues in specific proteins in blood and bones into gamma-carboxyglutamic acid (Gla), it is used to maintain calcium homeostasis, inhibit blood vessels Wall calcification, supports endothelial integrity, promotes bone mineralization, and is involved in tissue ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/53C12N15/60C12P7/66C12R1/125
CPCC12N9/1085C12N9/1022C12N9/1205C12N9/0006C12N9/88C12P7/66C12Y205/01074C12Y202/01007C12Y207/0103C12Y101/05003C12Y101/01261C12Y402/03003
Inventor 宋浩杨绍梅张国银蔡志刚
Owner TIANJIN UNIV MARINE TECH RES INST
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