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Promoter suitable for bacillus licheniformis and application of promoter in high-efficiency expression of target product

A Bacillus licheniformis promoter technology, applied in the field of genetic engineering and microbial metabolic engineering, can solve the problems of low expression efficiency and difficult expression, and achieve the effect of improving activity

Active Publication Date: 2020-11-13
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when Bacillus licheniformis expresses most foreign proteins, there are problems of difficult expression or low expression efficiency, and the promoter is identified as the main expression element for establishing high-efficiency gene expression tools. Preferred Strategies for Levels and Metabolites Metabolic Pathway Technology

Method used

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  • Promoter suitable for bacillus licheniformis and application of promoter in high-efficiency expression of target product
  • Promoter suitable for bacillus licheniformis and application of promoter in high-efficiency expression of target product
  • Promoter suitable for bacillus licheniformis and application of promoter in high-efficiency expression of target product

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Acquisition of promoters suitable for Bacillus licheniformis:

[0024] The promoter of the present invention is obtained by σ A and σ B Among the five binding sites recognized by the type factor, the core region of the promoter is guaranteed to be completely composed of σ A and σ B Under the premise of forming the recognition site of the type factor, four sites are selectively selected and chimerized. The nucleotide sequences of the five binding sites are respectively:

[0025] TTGACA...TATAAT;

[0026] GCCTTGA...CCCCAT;

[0027] TTCTCA...TAAAAT;

[0028] AAGCCA...TATATT;

[0029] GTAAAA...TATTAT;

[0030] According to the above 5 binding sites, after selecting four chimeric methods, four optimized promoters were artificially designed and synthesized, named R1-R4, and the corresponding nucleotide sequences are SEQ ID NO.2-SEQ Shown in ID NO.5.

Embodiment 2

[0032] Application of optimized promoter R1-R4 in increasing the expression of Bacillus licheniformis exogenous protein:

[0033] In this example, the R1-R4 promoter was directly introduced by primers, replacing the core region of the P43 promoter in the expression vector. The specific steps are as follows:

[0034] 1. Using the plasmid pHY-P43-GFP (DOI: 10.1016 / j.jbiotec.2020.02.015) constructed earlier in this experiment as a template, the vector backbone containing the optimized promoter was amplified by PCR using the primers in Table 1, and carried out DNA recovery;

[0035] Table 1: Primer design for expression vectors containing different optimized promoters

[0036]

[0037]

[0038] Note: The underline is the homology arm region, 18-19bp.

[0039] 2. Using DpnI enzyme to digest the remaining pHY-P43-GFP template;

[0040] 3. Use Vazyme’s homologous recombination enzyme (Exnase II) to treat the vector skeleton, and react at 37°C for 30 minutes to make the vecto...

Embodiment 3

[0048] The R2 promoter increases the expression of keratinase in Bacillus licheniformis Bright:

[0049] 1. Using pHY-R2-GFP and Bacillus licheniformis WX-02 genomic DNA as templates, the vector backbone pHY-R2 and keratinase were amplified by designing primers T5-F, T5-R, KER-F, and KER-R, respectively Gene ker fragment (shown in SEQ ID NO.6), and carry out DNA recovery;

[0050] 2. Use DpnI enzyme to digest the remaining pHY-R2-GFP template in the vector backbone pHY-R2;

[0051] 3. Use Vazyme's homologous recombination enzyme (Exnase II) to treat the purified vector backbone pHY-R2 and ker gene fragments, and react at 37°C for 30 minutes to connect the vector backbone and fragments; It was transformed into Escherichia coli; after finally being verified by sequencing, the recombinant plasmid (pHY-R2-KER) was obtained;

[0052] 4. Transfer the recombinant plasmid (pHY-R2-KER) into Bacillus licheniformis DW2, use tetracyclic antibiotics as selection markers, and then obtain ...

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Abstract

The invention relates to the field of gene engineering and microbial metabolism engineering, discloses promoters suitable for bacillus licheniformis and application of the promoters in efficient expression of a target product, and artificially designs four promoters (R1-R4) suitable for bacillus licheniformis. Then, green fluorescent protein GFP and keratinase KER are taken as target proteins, andcompared with a promoter P43, research finds that the promoter R2 has the best effect, the expression levels of GFP and KER are remarkably improved, the yield of GFP is improved by 63.5 times, and the enzyme activity of KER is improved by 7.8 times. Finally, the promoter R2 is applied to metabolic engineering, and compared with a control strain, the yield of poly gamma-glutamic acid and the yieldof bacitracin are increased by 52.09% and 20.39% respectively by applying the promoter R2 to metabolic pathway strengthening.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial metabolic engineering, in particular to a promoter suitable for bacillus licheniformis and its application in high-efficiency expression of target products. Background technique [0002] The promoter is a special DNA sequence located upstream of the gene transcription unit, which has the function of initiating gene transcription and plays an important role in regulating the level of gene transcription. With the development of synthetic biology and metabolic pathway engineering, people have gradually found that in order to achieve high yields of target products, it is crucial to enhance the expression level of target proteins and the metabolic flux of target metabolites. Since the promoter is the switch of gene transcription and is considered to be the main expression element for establishing high-efficiency gene expression tools, promoter modification is the preferred strategy for...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/50C12P21/00C12P13/02C12R1/10
CPCC07K14/32C07K14/43595C12N9/50C12P13/02
Inventor 陈守文饶忆蔡冬波马昕莫非
Owner HUBEI UNIV
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