Bacillus licheniformis for enhancing expression of cysP and preparation method and application thereof

A technology of Bacillus licheniformis and a construction method, which is applied in the field of genetic engineering bacteria transformation and can solve the problems of unpredictable influence of Bacillus licheniformis bacitracin synthesis and the like

Active Publication Date: 2020-06-19
LIFECOME BIOCHEM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current research has not yet resolved the mechanism of the sulfur transporter CysP in Bacillus licheniformis and whether it affects the supply of bacitracin precursor amino acids. The effect on bacitracin synthesis is also unpredictable

Method used

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  • Bacillus licheniformis for enhancing expression of cysP and preparation method and application thereof
  • Bacillus licheniformis for enhancing expression of cysP and preparation method and application thereof
  • Bacillus licheniformis for enhancing expression of cysP and preparation method and application thereof

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Experimental program
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Effect test

Embodiment approach

[0027] The specific embodiment of the construction method of Bacillus licheniformis that strengthens the expression of cysP is as follows:

[0028] 1. The specific operation steps of step (1) are:

[0029] According to the genome sequence of Bacillus subtilis 168, the upstream primer (cysp-P43-F) and downstream primer (cysp-P43-R) of the P43 promoter gene were designed, and the genome sequence of Bacillus subtilis 168 was used as a template to adopt the P43 promoter gene The upstream and downstream primers were used for PCR amplification to obtain the P43 promoter (305bp, such as figure 1 shown). At the same time, according to the genome sequence of Bacillus licheniformis DW2, the upstream primer (cysp-F) and downstream primer (cysp-R) of the sulfur transporter gene cysP were designed; The upstream and downstream primers of the transporter gene cysP were amplified by PCR to obtain the sulfur transporter gene cysP (1065bp, such as figure 1 shown); Meanwhile, according to the...

Embodiment 15

[0061] The difference between Example 15 and Example 14 is: the promoter in step (1) selects the PcysP promoter, and in step (1), the upstream primer and downstream primer of the PcysP promoter gene are designed with the Bacillus licheniformis DW2 genome sequence licheniformis DW2 genome sequence as a template, using the upstream and downstream primers of the PcysP promoter gene for PCR amplification to obtain the PcysP promoter, and the PcysP promoter fragment, the sulfur transporter gene cysP and the The amylase terminators are connected together in order to obtain the target gene fragments, the sequence of the target gene fragments is: PcysP promoter-sulfur element transporter gene cysP-amylase terminator, and the rest of the steps are the same as in Example 14, and finally Positive transformant, named: DW2 / pHY-cysP-1;

Embodiment 16

[0063] The difference between Example 16 and Example 14 is that the terminator in step (1) is selected as the promoter in step (1) and the PcysP promoter is selected, and in step (1) according to the Bacillus licheniformis DW2 genome sequence, design The upstream primer and downstream primer of the TcysP terminator gene, and according to the Bacillus licheniformis DW2 genome sequence, the TcysP terminator was obtained by PCR amplification using the upstream and downstream primers of the TcysP terminator, and the P43 promoter fragment, sulfur The element transporter gene cysP and the TcysP terminator are connected together in order to obtain the target gene fragment. The arrangement order of the target gene segment is: P43 promoter-sulfur element transporter gene cysP-TcysP terminator, and the remaining steps are the same as in Example 14 In the same way, a positive transformant was finally obtained, which was named: DW2 / pHY-cysP-2.

[0064] The Bacillus licheniformis DW2 / pHY-c...

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Abstract

The invention provides bacillus licheniformis for enhancing expression of cysP and a preparation method and application thereof. The bacillus licheniformis is prepared by transferring a plasmid carrier carrying a sulfur element transport protein gene cysP into bacillus licheniformis DW2, thereby realizing free expression of the sulfur element transport protein gene cysP in the bacillus licheniformis DW2, and achieving a purpose of enhancing the expression of the sulfur element transporter gene CysP. Compared with a strain obtained by directly transferring a blank plasmid vector without the cysP gene into the bacillus licheniformis DW2, the bacillus licheniformis for enhancing expression of cysP constructed by the invention has a large increase in bacitracin production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering bacteria transformation, in particular to a bacillus licheniformis that strengthens the expression of cysP and its preparation method and application. Background technique [0002] Bacillus licheniformis is recognized as an important industrial microbial strain with biosafety (GRAS), which widely exists in nature. In view of the characteristics of clear genetic background and high industrial application value of Bacillus licheniformis, it has been widely studied in recent years. Bacillus licheniformis is a Gram-positive bacterium, which is mainly used for the fermentation and production of poly-γ-glutamic acid, bacitracin, acetoin, 2,3-butanediol, lichenin and other biochemical products. Bacitracin is a class of cyclic peptide antibiotics composed of 12 amino acid residues, and its constituent amino acids include ornithine (Orn), D-phenylalanine (D-Phe), isoleucine (His), D-aspartic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/31C12N1/21C12P21/04C12R1/10
CPCC12N15/75C07K14/32C12P21/02C07K7/58
Inventor 陈守文蔡冬波李凌峰吴非李俊辉陈晓斌季潇炜楼丽君邱湘琪
Owner LIFECOME BIOCHEM
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