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Bacillus licheniformis expression host

A technology of Bacillus licheniformis and Bacillus subtilis, applied in the direction of bacteria, enzymes, biochemical equipment and methods, etc.

Active Publication Date: 2018-09-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No construction has been seen to simultaneously delete mpr (encoding extracellular metalloprotease), vpr (encoding serine protease), aprX (intracellular serine protease), epr (encoding micro extracellular protease), bpr (encoding Bacillus peptidase F), wprA (encodes cell wall-bound protease), aprE (encodes extracellular alkaline serine protease), bprA (encodes Bacillus peptidase F), hag (encodes flagellin), and amyL (encodes alpha-amylase) host in Bacillus licheniformis bacteria

Method used

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  • Bacillus licheniformis expression host
  • Bacillus licheniformis expression host
  • Bacillus licheniformis expression host

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Embodiment 1

[0054] Construction of embodiment 1 protease deletion strain BL10

[0055] 1. Annotation of Bacillus licheniformis WX-02 protease (or peptidase)

[0056] Using all the known and putative proteases or peptidases of Bacillus licheniformis listed in the MEROPS database, Blast found the corresponding protease or peptidase genes in the WX-02 genome sequence, and then predicted them with Signal-3L and Cell-PLoc Its location in the cell, extracellular (including cell wall), intracellular or cell membrane. Then, using the reported extracellular proteomics results of B. licheniformis as a reference, some proteases or peptidases that can be secreted extracellularly without signal peptides were corrected, and their protein molecular weights were calculated using EditSeq. Through the analysis of the B. licheniformis genome sequence obtained by sequencing, it is concluded that there are 166 known and putative proteases (or peptidases) in the genome, among which the (known and putative) pr...

Embodiment 2

[0075] High-efficiency secretory expression of embodiment 2α-amylase

[0076] The construction process of B. licheniformis α-amylase expression plasmid pP43SAT is as follows: Figure 8 shown. A pair of primers P43F and P43R1 were designed according to the sequence of the P43 promoter on the B.subtillis168 genome published by NCBI,

[0077] P43F: 5'-GGAATTCTGATAGGTGGTATGTTTTCG-3' (EcoRI)

[0078] P43R1: 5'-AAGCCGTTTTTGTTGTTTCATTTCATGTGTACATTCCTCTC-3'

[0079] The promoter P43 fragment was amplified by PCR with a size of 305bp.

[0080] According to the amyL gene sequence (see SEQ ID NO.9) in the whole genome sequence of B. licheniformis WX-02 measured in this experiment, the accession number on NCBI is MUY_00877, and a pair of primers PamyLF1 and PamyTR were designed,

[0081] PamyLF1:

[0082] 5'-GAGAGGAATGTACACATGAAATGAAACAACAAAAACGGCTT-3'

[0083] PamyTR: 5'-GAAGATCT CGCAATAATGCCGTCGCACTG-3'(BglII)

[0084] Using the genomic DNA of B. licheniformis WX-02 as a template...

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Abstract

The invention discloses a bacillus licheniformis host bacterium BL10 with multiple gene deletion, the accession number of which is CCTCCNO:M2013400. The host bacterium derives from a bacillus licheniformis WX-02 which partially or completely has deletion of ten genes. The ten genes comprises eight protease genes (mpr encoding a metalloprotease; vpr encoding a serine protease; aprX encoding an intracellular serine protease; epr encoding a minimal extracellular protease; bpr encoding a bacillus peptidase F; wprA encoding a protease combined with cell walls; aprE encoding an extracellular alkaline serine protease; and bprA encoding a bacillus peptidase F) and two extracellular secretory protein genes (hag encoding flagellin; and amyL encoding alpha-amylase). The BL10 has completely no extracellular protease activity and can reduce the degradation effect of a protease on a target protein. When the target protein is expressed by the expression host, the expression level is higher, and the host bacterium benefits protein expression enhancement.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a recombinant bacillus licheniformis BL10 host strain and a method for expressing a target protein in the host cell. Background technique [0002] In the process of studying protein expression, a variety of valuable expression systems have been developed, mainly including prokaryotic expression systems and eukaryotic expression systems. The prokaryotic expression system was the first to be adopted, and it is also a relatively mature expression system that has been studied at present. At present, the most commonly used prokaryotic expression systems include Escherichia coli expression system, Bacillus subtilis expression system and Streptomyces expression system, etc. Among them, Escherichia coli expression system is the most widely used, and Bacillus subtilis is used to secrete and express target proteins. More and more attention has been paid to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/26C12N9/20C12N9/50C12N15/75C12R1/10
Inventor 陈守文周银华魏雪团陈敬帮祁高富冀志霞
Owner HUAZHONG AGRI UNIV
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