Bacillus subtilis with inactivated extracellular protease as well as construction method and application of bacillus subtilis

A Bacillus subtilis and extracellular protease technology, applied in the field of genetic engineering, can solve the problems of unfavorable recombinant protein secretion and expression, loss of target protein yield, etc., and achieve good amino acid utilization ability, no nutritional deficiency, and improved transformation efficiency

Active Publication Date: 2022-01-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extracellular proteases have a high tendency to degrade heterogeneous secreted proteins, which will lead to a serious loss of target protein production, which is not conducive to the secretory expression of recombinant proteins

Method used

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  • Bacillus subtilis with inactivated extracellular protease as well as construction method and application of bacillus subtilis
  • Bacillus subtilis with inactivated extracellular protease as well as construction method and application of bacillus subtilis
  • Bacillus subtilis with inactivated extracellular protease as well as construction method and application of bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of CRISPR series plasmids

[0049] As described by Wu et al. in CAMERS-B: CRISPR / Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering2020, 117:1817–1825, the CRISPR / Cpf1 system consists of two plasmids pHT-XCR6 and pcrF19NM2 (Available through the molecularcloud plasmid sharing platform, numbers MC_0068418 and MC_0101256, respectively). Plasmid pHT-XCR6 (ampicillin resistance in Escherichia coli, chloramphenicol resistance in Bacillus subtilis) is a Cpf1 expression vector, in which Cpf1 is induced and regulated by xylose; in addition, the plasmid also contains NgAgo protein Genes used to improve the efficiency of homologous recombination during gene editing. Plasmid pcrF19NM2 (both kanamycin resistant in Escherichia coli and Bacillus subtilis), and a temperature-sensitive plasmid in Bacillus subtilis, cannot replicate when cultured above 37°C, and is used as a crRNA expression vector ...

Embodiment 2

[0078] Embodiment 2: Construction of protease inactivated bacterial strain B.subtilis G601

[0079] The Cpf1 protein expression plasmid pHT-XCR6 of the CRISPR / Cpf1 system was transformed into a competent Bacillus subtilis, and spread on an LB plate containing chloramphenicol resistance until a single colony grew out. Then the bacterial strain transformed with pHT-XCR6 was made into a competent state, and transformed into the pcrF19-epr-xcomKS constructed in Example 1. After the plasmid was added to the competent state and cultivated for two hours, the plate was not directly coated, but the bacterial liquid Centrifuge (4000rmp, 2min) and resuspend in 500μL LB containing chloramphenicol, kanamycin and 3% xylose for overnight culture, then centrifuge and concentrate to 150μL the next day and add chloramphenicol and kanamycin LB plates with plain and 3% xylose. After a single colony grows, colony PCR can be performed to verify whether the gene editing has been completed. The suc...

Embodiment 3

[0080] Example 3: Verification of protease inactivated strain B.subtilis G601

[0081] Such as image 3 As shown, the trpC0 on B. subtilis G601 was amplified and sequenced, which was consistent with the sequence of the designed mutation. Such as Figure 4 As shown, the wild-type strain B.subtilis 168 before mutation ( Figure 4 Upper) cannot grow on synthetic media, the mutated B.subtilis G601 ( Figure 4 Below) can grow normally. Such as Figure 5 As shown, put the gudB on the B.subtilis G601 + Amplified sequencing, which is consistent with the sequence of the designed mutation. Such as Figure 6 As shown, the engineered strains were subjected to colony PCR using gene name-VF and gene name-VR, and the results showed that B. subtilis G601 had integrated the xylose-inducible promoter P xylA The controlled transcription factor comK-comS gene was back-mutated to trpC0, and six extracellular protease genes were knocked out. Such as Figure 7 As shown, the ability of the ...

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Abstract

The invention discloses bacillus subtilis with inactivated extracellular protease as well as a construction method and application the bacillus subtilis. B.subtilis 168 is taken as an original strain, a transcription factor comK-comS gene controlled by a xylose inducible promoter PxylA is integrated on a genome by using a CRISPR/Cpf1 technology, so that the transformation efficiency of the bacillus subtilis is greatly improved; a trpC gene and a gudB gene are subjected to reverse mutation, so that tryptophan can be synthesized, and glutamic acid family amino acids can be effectively utilized; and besides, six extracellular protease genes are knocked out, so that the metabolic pressure of cells is reduced, and recombinant proteins can be effectively accumulated outside the cells. The content of extracellular sfGFP produced when a final engineering strain G601 is used for fermentation is 1.88 times of that of the B.subtilis 168, and the total content of intracellular and extracellular sfGFP is 1.94 times of that of the B.subtilis 168. An engineering bacterium constructed by the invention is simple and convenient to transform, has no nutritional deficiency, has better amino acid utilization capacity and low extracellular protease activity, can be widely applied to secretory expression of a recombinant protein, and has a wide application prospect.

Description

technical field [0001] The invention relates to a Bacillus subtilis with inactivated extracellular protease and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Recombinant protein refers to the use of recombinant DNA technology to optimize and modify the gene encoding the target protein, use a certain vector to introduce the target gene into an appropriate host cell, express the target protein, and obtain a biologically active protein through extraction and purification techniques. products. Recombinant proteins are widely used in the fields of food and medicine, and have produced huge economic value and social benefits. In order to achieve high-efficiency expression of recombinant proteins, the transformation and optimization of expression systems is a hot spot and focus of current research. However, the use of microbial expression systems for heterologous protein production to produce recombi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/67C12N15/57C12N15/31C12N15/53C12N15/90C12R1/125
CPCC12N9/54C07K14/32C12N9/0016C12N15/75C12N15/902C12Y304/21062C12Y104/01002C12Y304/24028
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰李洋武耀康
Owner JIANGNAN UNIV
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