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Method for transforming bacillus subtilis FMMs and application thereof

A Bacillus subtilis, domain technology, applied in the field of transforming Bacillus subtilis FMMs, can solve problems such as lysis and severe cells, and achieve the effects of simple construction method, good application prospects, and easy use.

Active Publication Date: 2019-10-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, severe cell lysis often occurs in the later stage of fermentation, which ultimately inhibits the efficient synthesis of products.

Method used

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  • Method for transforming bacillus subtilis FMMs and application thereof
  • Method for transforming bacillus subtilis FMMs and application thereof
  • Method for transforming bacillus subtilis FMMs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The transformation and verification of the FMMs of embodiment 1 Bacillus subtilis

[0037] On the basis of Bacillus subtilis BSGN6, the yisp homologous recombination integration expression frame is integrated at the tcyp or yhcm site on the Bacillus subtilis BSGN6 genome. Use integration site tcrp sequence (length 1kb), spectinomycin resistance gene spc sequence (nucleotide sequence shown in SEQ ID NO.5, including lox site), yisp gene sequence, yhcM downstream sequence (length 1kb) Build the integration box. Through homologous recombination, the integration frame obtained above was integrated into the genome of Bacillus subtilis BSGN6. Through spectinomycin resistance plate screening, colony PCR verification, and sequencing, it was confirmed that the integration was successful and the recombinant Bacillus subtilis overexpressing yisp was obtained. The SPFH gene fragment was inserted into the multiple cloning site of the pP43NMK plasmid using Gibson seamless assembly t...

Embodiment 2

[0039] Embodiment 2 analyzes the impact of FMMs remodeling on the growth of Bacillus subtilis cells

[0040] Cultivate the transformant of BSGN6-pP43 and BSGFMM1 recombinant Bacillus subtilis in seed medium (10g / L peptone, 5g / L yeast powder and 10g / L sodium chloride) for 8-10h, then follow the inoculum size of 2-5% Transfer to fermentation medium (40g / L glucose, 6g / L peptone, 12g / L yeast powder, 6g / L ammonium sulfate, 12.5g / L dipotassium hydrogen phosphate, 2.5g / L potassium dihydrogen phosphate, 5g / L carbonic acid Calcium 10ml / L trace elements), cultured in a 250mL shake flask at 37°C and 220rpm for 72h.

[0041] Use UV-visible spectrophotometer to regularly measure the absorbance value OD of fermentation broth 600 To characterize cell growth, such as figure 2 Therefore, the OD of BSGN6-pP43 and BSGFMM1 strains within 45h 600 There was no significant difference in the value, after 45h, the OD of BSGFMM1 600 The value was significantly higher than that of BSGN6-pP43 strain...

Embodiment 3

[0042] Example 3 Construction of recombinant Bacillus subtilis BSGFMM1-AY

[0043] On the basis of Bacillus subtilis BSGFMM1, the fusion gene expression cassette of SPFH and yqaB was integrated at the hprK site of Bacillus subtilis BSGFMM1 genome. Using the integration site hprK sequence (length 1kb), the chloramphenicol resistance gene CmR sequence (the nucleotide sequence is shown in SEQ ID NO.6, including the lox site), the C-terminal SPFH gene sequence with GS linker ( 0.68kb), yqaB gene sequence (0.57kb), nagR gene and its downstream gene sequence (length 1kb) to construct the SPFH-yqaB fusion gene integration frame, wherein, SPFH and yqaB are fusion expressions, and the linker nucleotides connected between the two The acid sequence is shown in SEQID NO.4. The integration frame obtained above was integrated into the genome of Bacillus subtilis BSGFMM1 through homologous recombination. Through chloramphenicol resistance plate screening, colony PCR verification, and seque...

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Abstract

The invention discloses a method for transforming bacillus subtilis FMMs and application thereof, and belongs to the technical field of genetic engineering. According to the method, the ratio of FMMson a plasma membrane is remarkably increased by strengthening the sterol analogue precursor synthetase, namely squalene synthase and scaffold protein. Compared with a control strain BSGN6-AY, the recombinant bacillus subtilis BSGFMM1-AY constructed through the method can immobilize more pathway enzyme required by N-acetylglucosamine in FMMs to form a substrate channel, and the catalytic efficiencyof the enzyme is remarkably improved. In a shake flask fermentation process of a composite culture medium, the GlcNAc titer of the control strain BSGN6-AY is only 2.84 g / L, and the GlcNAc titer of the BSGFMM1-AY is increased to 8.86 g / L. The construction method of the recombinant bacillus subtilis is simple, high in universality and convenient to use, and has good application prospects.

Description

technical field [0001] The invention relates to a method for transforming Bacillus subtilis FMMs and an application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Membrane rafts are a type of densely structured microdomains rich in cholesterol and other substances on the plasma membrane. They are rich in long-chain saturated fat chains and have low fluidity, with a size of about 70nm. In addition to the above-mentioned components, membrane rafts are also rich in some scaffolding proteins. These proteins usually contain some special domains, such as the SPFH (Stomatin-Prohibitin-Flotillin-HflC / K) domain, not only functionally dependent on membrane rafts , but also affect the formation and stability of membrane rafts. Membrane rafts can provide a platform for protein aggregation to facilitate the interaction between proteins, and the microenvironment of membrane rafts is conducive to correct protein folding and effective allost...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62C12N15/75C12P19/26C12R1/125
CPCC07K14/00C07K2319/00C12N9/1085C12P19/26C12Y205/01021
Inventor 刘龙吕雪芹张成金柯李梦莹李江华堵国成陈坚
Owner JIANGNAN UNIV
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