Recombinant bacterium for promoting bacillus subtilis to synthesize menadione-7 and gene modification method thereof

A technology of Bacillus subtilis and menadione, applied in the field of biological genetic engineering, can solve the problems of organic solvent residue, low yield, dangerous production environment and the like

Pending Publication Date: 2020-08-21
TIANJIN UNIV MARINE TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the process of producing MK-7 by chemical synthesis is constantly innovating, the defects of low yield, many by-products, residual organic solvents, dangerous production environment, and hidden dangers in environmental protection have not been completely resolved, which restricts the production and development of MK-7. application

Method used

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  • Recombinant bacterium for promoting bacillus subtilis to synthesize menadione-7 and gene modification method thereof
  • Recombinant bacterium for promoting bacillus subtilis to synthesize menadione-7 and gene modification method thereof
  • Recombinant bacterium for promoting bacillus subtilis to synthesize menadione-7 and gene modification method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This embodiment provides a genetic modification method for promoting the synthesis of menadione-7 by Bacillus subtilis, specifically as follows:

[0023] 1. Materials

[0024] Strains, Plasmids and Media

[0025] See Table 1 for details of all strains and plasmid information involved in the present invention.

[0026] 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 O 0.5g / L, pH7.3.

[0027] The strains and plasmids involved in the experiments in Table 1

[0028]

[0029]

[0030] Reagents and instruments

[0031] FastTaq enzyme, Hifi DNA polymerase, and dNTP were all purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the standard product MK-7 was purchased from Chroma...

Embodiment 2

[0044] The cultivation of embodiment 2 recombinant bacterial strain BSMK_11

[0045] Shake flask fermentation culture and determination of cell growth, fermentation medium and fermentation conditions are shown in Table 3.

[0046] Table 3 fermentation medium and fermentation conditions

[0047] parameter scope glycerin 20~80mL / L soy peptone 60~180g / L Yeast extract 0~20g / L K 2 HPO 4

1~5g / L MgSO 4 ·7H 2 o

0.1~0.8g / L pH 6.5～7.5 Inoculation amount 1％～6％ temperature 35～45℃ Rotating speed 100~250r / min fermentation time 72-144h

[0048] 500mL shake flask fermentation: Pick a single colony on a newly activated plate and insert it into a 250mL shake flask filled with 30mL LB medium, shake and culture at 200r / min, 37°C for 14h; In the 500mL Erlenmeyer flask (three parallel), 37 ℃, dark conditions, 220r / min shaking culture for 120h.

[0049] Biomass measurement: take samples after 6 hours of f...

Embodiment 3

[0060] Embodiment 3 carbon source optimization

[0061] It can be seen from Table 4 that the growth of the starting bacterium BSMK_9 decreased at 48 hours of fermentation, and then decreased, that is, secondary growth phenomenon appeared, indicating that the carbon source glycerol was exhausted at this time, and then soybean peptone provided nitrogen source and nitrogen source. Carbon source to meet the growth requirements of Bacillus subtilis. In order to further increase the yield of MK-7, the present invention adds 1.0% glycerol, glucose, sucrose, raffinose, sorbitol and mannitol to the fermentation medium respectively. As can be seen from Table 6, the addition of glycerol causes the MK-7 yield to decrease by 8.3% compared with the initial condition, indicating that the presence of 3.0% glycerol in the fermentation medium is most conducive to the synthesis of MK-7; in addition, the addition of glucose, raffinose or mannitol Both also resulted in a decrease in the yield of ...

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Abstract

The invention discloses a gene modification method for promoting bacillus subtilis to synthesize menadione-7. The gene modification method comprises the following steps: (1) constructing an original strain BSMK_9; and (2) carrying out overexpression of the vitreoscilla hemoglobin gene vgb. According to the invention, BSMK_9 derived from bacillus subtilis 168 is used as an original strain, and by overexpression of vitreoscilla hemoglobin, transmission of intracellular oxygen in cells is promoted, the utilization efficiency of oxygen is improved, and the influence on MK-7 synthesis is investigated; the maximum synthesis amount of MK-7 is promoted by optimizing carbon source combination and tank fermentation, and a theoretical basis is provided for construction of an MK-7 high-yield strain bygenetic modification and fermentation optimization of bacillus natto in the future.

Description

Technical field: [0001] The invention relates to a genetic modification method for promoting the synthesis of menadione-7 by Bacillus subtilis and its recombinant bacteria, belonging to the field of biological genetic engineering. Background technique: [0002] Natural fat-soluble vitamin K includes plant-derived vitamin K1 (also known as phylloquinone, PK) and bacterial-derived vitamin K2 (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, pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12P7/66C12R1/125
CPCC07K14/805C12P7/66
Inventor 宋浩杨绍梅张国银蔡志刚
Owner TIANJIN UNIV MARINE TECH RES INST
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