Chassis strain for producing alkaline protease as well as construction method and application of chassis strain

A construction method and protease technology are applied in the breeding of industrial microorganisms, the chassis strain for producing alkaline protease and its construction field, which can solve problems such as lifting, unfavorable secretion and purification of target products, and high risk of contamination.

Pending Publication Date: 2022-01-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only will it lead to a higher risk of bacterial contamination, it is not conducive to the secretion and purification of the target product, but it will also increase the cost of industrial operations and affect the improvement of economic benefits

Method used

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  • Chassis strain for producing alkaline protease as well as construction method and application of chassis strain
  • Chassis strain for producing alkaline protease as well as construction method and application of chassis strain
  • Chassis strain for producing alkaline protease as well as construction method and application of chassis strain

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0036]According to a preferred embodiment of the present invention, the six extracellular protease genes on the Bacillus amyloliquefaciens genome aprE, bpr, vpr, mpr, nprE, epr, exopolysaccharide gene cluster eps, polyglutamic acid gene cluster The phage-related genes whose pgs and nucleotide sequence are shown in SEQ ID NO: 1 were knocked out. The way of knockout can adopt conventional means in this field, for example, by means of homologous recombination, construct a knockout vector and electrotransform into Bacillus amyloliquefaciens, and knock out the above-mentioned genes from the genome through single and double exchange, preferably, The knockout vector is pWH-T2 plasmid containing Kana resistance gene.

[0037] According to the present invention, the method of heterologous overexpression of the above-mentioned genes can adopt conventional means in the art, for example, the expression cassette of the alkaline protease gene aprE is inserted into the genome of Bacillus amy...

specific Embodiment approach

[0043] According to a specific embodiment of the present invention, the construction method includes: knocking out six extracellular protease genes aprE, bpr, vpr, mpr, nprE, and epr in the starting strain Bacillus amyloliquefaciens, to obtain strain BAΔ6; further knocking out The extracellular polysaccharide gene cluster eps was removed to obtain the strain BAΔ6Δeps; the polyglutamic acid gene cluster pgs was further knocked out to obtain the strain BAΔ6ΔepsΔpgs; the phage-related gene 3049-3052 was further knocked out to obtain the strain BAΔ6ΔepsΔpgsΔ3049-3052.

[0044] Further, the expression vector containing the expression cassette of the alkaline protease gene aprE was transformed into the strain BAΔ6ΔepsΔpgsΔ3049-3052 to obtain Bacillus amyloliquefaciens BAΔ6ΔepsΔpgsΔ3049-30521 with high production of alkaline protease.

[0045] According to a more preferred embodiment of the present invention, the construction method includes the following steps:

[0046] (1) Knock ou...

Embodiment 1

[0081] Embodiment 1: the construction of genetic engineering strain

[0082] (1) Knock out the target gene

[0083] 1) Amplify the homologous sequence of the target gene

[0084] According to the genome data of Bacillus amyloliquefaciens CGMCC No.11218, it was designed and amplified by PCR to obtain the UP end and DOWN end of the homology arm sequence at both ends of the target gene sequence. Target gene-DOWN-F, target gene-DOWN-R two sets of primers (see the primer table), and use the CGMCC No.11218 genome as a template for PCR amplification; the amplification reaction system is as follows:

[0085] Primer F 2μL Primer R 2μL DNA template 2μL PrimerStar Enzyme 25 μL ddH2O 19μL

[0086] The amplification program was set as follows: pre-denaturation: 95°C for 5 min; denaturation: 95°C for 30 s; annealing: 56°C for 45 s; extension: 72°C for 5 s; reaction for 30 cycles; extension: 72°C for 10 min.

[0087] The PCR product was subjected ...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to breeding of industrial microorganisms, in particular to a chassis strain for producing alkaline protease as well as a construction method and application of the chassis strain. The invention provides a bacillus amyloliquefaciens gene engineering strain, which does not express six extracellular protease genes aprE, bpr, vpr, mpr, nprE and epr on a bacillus amyloliquefaciens genome, an extracellular polysaccharide gene cluster eps, a polyglutamic acid gene cluster pgs and a bacteriophage related gene with a nucleotide sequence as shown in SEQ ID NO: 1, and preferably, the bacillus amyloliquefaciens gene engineering strain also expresses an alkaline protease gene aprE in a heterologous overexpression manner. According to actual conditions in experiments and production, adverse factors influencing fermentation performance are analyzed, the chassis strain for producing alkaline protease at high yield is finally obtained through transformation and gene simplification of a genetic engineering host, and a new thought is provided for construction of the chassis strain for producing the alkaline protease at high yield.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a chassis strain for producing alkaline protease and its construction method and application. Background technique [0002] Among many industrial enzymes, alkaline protease mainly produced by microorganisms is a type of enzyme that can hydrolyze protein peptide bonds under alkaline conditions, and its optimum pH is generally 9-11. It is widely used and researched in manufacturing, textile manufacturing and other industries. [0003] Along with the continuous growth of market demand for alkaline protease, people begin to focus on the construction of the high-yield chassis strain of alkaline protease. Escherichia coli (E.coli) and Pichia pastoris (Pichia pastoris) are used as the preferred host strains for the high-efficiency expression of alkaline protease, but the yield is low and the stability is poor, which is dif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/57C12N15/31C12N15/75C12N9/54C12R1/07
CPCC12N9/54C12Y304/21014C12N15/75C07K14/32C12N2800/60
Inventor 李玉路福平刘逸寒李昕悦齐威史超硕李庆刚
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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