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Method for increasing yield of Menaquinone-7 (MK-7) by strengthening functional membrane microdomains (FMMs) of bacillus subtilis

A technology of Bacillus subtilis and menadione, which is applied in the field of metabolism, can solve the problems of increasing the proportion of FMMs and increasing the storage space of MK-7, and achieve the effect of increasing production

Active Publication Date: 2021-12-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No studies have yet shown whether it is possible to enhance the FMMs in Bacillus subtilis, increase the proportion of FMMs in the plasma membrane, and increase the storage space of MK-7 on the plasma membrane to increase the production of MK-7

Method used

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  • Method for increasing yield of Menaquinone-7 (MK-7) by strengthening functional membrane microdomains (FMMs) of bacillus subtilis
  • Method for increasing yield of Menaquinone-7 (MK-7) by strengthening functional membrane microdomains (FMMs) of bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Construction of recombinant Bacillus subtilis BSQ1, BSQ2

[0026] The P43 promoter was integrated into the upstream of floA (yuaG) and floT (yqfB) on the recombinant Bacillus subtilis BS20 genome through the cre / loxp system to construct recombinant Bacillus subtilis BSQ1 and BSQ2, and the specific construction process was as follows.

[0027] By overlap extension PCR, the following sequences were amplified. The amplified sequences required for the P43-floA integration frame were the upstream sequence of floA (length 1000bp, sequence such as SEQ ID NO.1), the chloramphenicol resistance gene zeo sequence ( 1309bp, sequence such as SEQ ID NO.2), P43 promoter (0.3bp, sequence such as SEQ ID NO.3) and floA sequence (996bp, sequence such as SEQ ID NO.4). The amplified sequence required for the P43-floT integration frame is the floT upstream sequence (length 1000bp, sequence such as SEQ ID NO.5), the chloramphenicol resistance gene zeo sequence (1309bp), the P43 ...

Embodiment 2

[0032] Embodiment 2: Construction of recombinant Bacillus subtilis BSQ12

[0033] On the basis of BSQ1 obtained in Example 1, a method similar to Example 1 was used to integrate the P43 promoter upstream of floT on the BSQ1 genome to construct recombinant Bacillus subtilis BSQ12.

[0034] The floT constructed in Example 1 up The -lox71-zeo-lox66-p43-floT fusion expression cassette was transferred into BSQ1 by chemical transformation, screened on a chloramphenicol resistance plate, colony PCR verification, sequencing, transferred to a Cre plasmid, and screened on a Kanna resistance plate. IPTG induced expression, Cre plasmid elimination, spot plate verification, and finally obtained recombinant Bacillus subtilis BSQ12 integrating P43-floA and P43-floT expression cassettes.

Embodiment 3

[0035] Embodiment 3: bacterial strain fermentation produces MK-7

[0036] (1) Preparation of seed solution

[0037] Pick a single colony of recombinant Bacillus subtilis BS20, and BSQ1, BSQ2, and BSQ12 constructed in Examples 1 and 2, and inoculate them into 15mL shaking tubes, each containing 2mL liquid seed medium, at 37°C and 220rpm Shaker culture 10h. BS20 was used as a control, and each strain was replicated three times.

[0038] (2) Fermentation culture

[0039] Inoculate the seed liquid obtained in step (1) into a 250mL Erlenmeyer flask according to the inoculation amount of 10%. Each bottle is equipped with 20mL of fermentation medium, cultivated on a shaking table at 41°C and 220rpm for 3 days, and took samples every 24h for OD 600 detection and sample preparation. The specific method is as follows.

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Abstract

The invention discloses a method for increasing yield of Menaquinone-7 (MK-7) by strengthening functional membrane microdomains (FMMs) of bacillus subtilis. According to the method disclosed by the invention, a scaffold protein FloA in the bacillus subtilis FMMs for producing the MK-7 is expressed in a strengthened manner. According to the method, the scaffold protein in recombinant bacillus subtilis BS20 with high yield of MK-7 is strengthened by using a powerful promoter P43, and after the proportion of the FMMs in a cytoplasmic membrane is increased, the yield of the MK-7 is further increased; and compared with a control strain BS20, the yield of the recombinant bacillus subtilis BSQ1 with strengthened FloA is the highest, and after fermentation is carried out for 6 days, the yield of the BSQ1 strain reaches 417.08mg / L and is increased by 16.57% compared with that of the BS20 strain.

Description

technical field [0001] The invention relates to a method for increasing the yield of menadione by strengthening the functional membrane micro-domain of Bacillus subtilis, and belongs to the field of metabolism technology. Background technique [0002] As an important fat-soluble vitamin, vitamin K is one of the indispensable important vitamins for the human body. It plays a key role in accelerating blood coagulation, preventing cardiovascular sclerosis and treating osteoporosis. Vitamin K uses 2-methyl-1,4-naphthoquinone as the backbone, and vitamin K can be divided into different subtypes according to the structure of the branched chain at the C3 position. Among them, menaquinone-7 (MK-7) has attracted much attention in the fields of functional food and medicine due to its long half-life and high affinity in the human body. The Chinese patent title CN110157749B applied by the inventor's research group in the early stage has achieved a relatively high MK-7 production in Bac...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N15/31C12P7/66C12R1/125
CPCC12N15/75C07K14/32C12P7/66
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰董雅君金柯张智航王凌锐
Owner JIANGNAN UNIV