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Bacillus subtilis natto recombinant bacteria and construction method and application thereof

A technology of Bacillus natto and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of unsuitability for industrial production, low concentration of vitamin K2, low concentration of γ-polyglutamic acid and the like

Active Publication Date: 2019-08-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Bacillus subtilis natto (Bacillus subtilis natto) ND-1-A27 has a low concentration of vitamin K2 (MK-7) and low concentration of γ-polyglutamic acid in industrial fermentation medium, so it is not suitable for industrial use. Production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Preparation method of non-resistant Bacillus natto ND-1-A27A△ubiA:

[0030] (1) Design and synthesize the Cas9 protein expression cassette (SEQ ID No.6), sgRNA expression cassette (SEQ ID No.7), and sgRNA fragment (SEQ ID No.8) that meet the codon characteristics of Bacillus natto ND-1-A27. );

[0031] The synthesized Cas protein expression box T-A was cloned from pMD19-T-simple vector to obtain plasmid T-Cas9. Then use primers to amplify the fragments, and after gel recovery, seamlessly splice into pMA5 digested with BamH I and Mlu I to obtain plasmid pMAC;

[0032] (2) Use primers to amplify the sgRNA designed with ubiA as the target, and use Primer star DNA polymerase to amplify the sgRNA scaffold expression frame with target N20;

[0033] After purification, the amplified product is digested with MluI and then ligated into the MluI site of plasmid pMA5-Cas9;

[0034] (3) Extract the genomic DNA of Bacillus natto ND-1-A27, and use Primer Star DNA polymerase and related prime...

Embodiment 2

[0039] Preparation method of Bacillus natto ND-1-A27ACD△ubiA△aroH:

[0040] (1) Use primers to amplify the sgRNA designed with aroH as the target, and use Primer star DNA polymerase to amplify the sgRNA scaffold expression cassette with the target N20;

[0041] After purification, the amplified product is digested with MluI and then ligated into the MluI site of plasmid pMA5-Cas9;

[0042] (2) Extract the genomic DNA of Bacillus natto ND-1-A27, and use Primer Star DNA polymerase and related primers to amplify each 500bp of the upstream and downstream of aroH, as well as the menC and menD genes as homologous repair sequences, and follow the 500bp upstream of aroH , MenC, menD and 500bp downstream of aroH are seamlessly spliced ​​into the pst I site of plasmid pMA5-Cas9. Obtain the Bacillus natto ND-1-A27 genome editing vector pMAC-1;

[0043] (3) Insert plasmid DNA into the prepared Bacillus natto ND-1-A27A△ubiA competent cells; in the electrotransformation competent cells, ice bath f...

Embodiment 3

[0047] The application of using metabolically modified strains to jointly produce vitamin K2 (MK-7) and γ-polyglutamic acid. It includes the following steps:

[0048] (1) Preparation of liquid cell culture of Bacillus natto ND-1-A27ACD△ubiA△aroH: pick a loop of Bacillus natto ND-1-A27ACD△ubiA△aroH strain on nutrient agar medium and inoculate it in the cell In a 250mL Erlenmeyer flask with 30mL seed culture medium, place it on a shaker at 37°C and cultivate for 8h to logarithmic phase at a speed of 120r / min to obtain Bacillus natto ND-1-A27ACD△ubiA△aroH Cell liquid culture of the strain.

[0049] (2) The composition and ratio of the fermentation medium are: glycerol 50g / L, soy peptone 30g / L, yeast powder 0.6g / L, K 2 HPO 4 0.3g / L; sterilize at 121°C for 20 minutes.

[0050] (3) Shake flask fermentation: The cell liquid culture of the above Bacillus natto ND-1-A27ACD△ubiA△aroH strain was inoculated into a sterilized 30mL fermentation culture at an inoculum of 2% (w / w) In a 250mL Erle...

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Abstract

The invention discloses bacillus subtilis natto recombinant bacteria and a construction method and application thereof, and belongs to the technical field of genetic engineering. According to the system, it is the first attempt to utilize a CRISPR-CAS gene editing technology for knockout of key enzymes ubiA and aroH in a by-product feedback metabolic pathway, expression of key enzyme genes menA, menC and menD is enhanced on genomes, gamma-polyglutamic acid is obtained while the concentration of vitamin K2(MK-7) is increased, the concentration of the gamma-polyglutamic acid reaches a high level, and a new idea of combined production of the vitamin K2(MK-7) and the gamma-polyglutamic acid is provided.

Description

Technical field [0001] The invention relates to a recombinant Bacillus natto bacteria, a construction method and application thereof, and belongs to the technical field of genetic engineering. Background technique [0002] Vitamin K 2 Menaquinone (MK) is a fat-soluble vitamin, a derivative of the naphthoquinone group with phylloquinone biological activity, and one of the indispensable important vitamins in the human body. Vitamin K2 is a series of menadione compounds, light yellow crystals. There are 14 forms according to the length of the isoprene side chain at the C-3 position on its molecular structure. MK-n means isoprene on the side chain. The number of units), of which MK-7 (natural vitamin K2) has the most significant biological activity. [0003] γ-Polyglutamic acid (γ-PGA) is a kind of multifunctional biopolymer polymerized by D-glutamic acid and (or) L-glutamic acid monomer through γ-amide bond, γ- PGA is the main component of natto viscous colloid and has the effect of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/113C12N15/75C12N15/90C12P7/66C12P13/02C12R1/125
CPCC12N9/1022C12N9/1085C12N9/88C12N9/90C12N15/113C12N15/75C12N15/902C12P7/66C12P13/02C12Y202/01009C12Y402/01113
Inventor 许正宏史劲松李会张晓梅徐行刘珍
Owner JIANGNAN UNIV
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