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An engineering strain and its fermentation process for highly expressing mccj25

A high-efficiency expression and fermentation technology, applied in fermentation, genetic engineering, bacteria, etc., can solve the problem of low expression of MicrocinJ25, and achieve the effect of high-efficiency expression and simple post-processing purification process.

Active Publication Date: 2022-06-28
安杰利(重庆)生物科技有限公司
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Problems solved by technology

[0009] This solution provides an engineering strain that highly expresses MccJ25 to solve the problem of low expression of Microcin J25 in the prior art

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  • An engineering strain and its fermentation process for highly expressing mccj25
  • An engineering strain and its fermentation process for highly expressing mccj25
  • An engineering strain and its fermentation process for highly expressing mccj25

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Embodiment Construction

[0031] The following is a further detailed description through specific embodiments:

[0032] Part 1: Construction of an engineered strain highly expressing Microcin J25

[0033] The engineered strain is constructed by the following methods: performing gene manipulation on mcjABCD respectively to become one direction, and sharing a promoter T7 in E. coli, cloned in the vector pBR322, and realizing high-efficiency expression in E. coli. The expression amount is 2.4g / L;

[0034] At the same time, error-prone PCR was used for directed evolution, and mcjABCD was mutated, and the amino acid sequence was changed. When expressed in E. coli, the expression and antibacterial activity of the product were significantly increased, and the expression amount was 4.1 g / L; Expression, shared a promoter PslpA, cloned in pLEM415 vector, the expression amount was 2.3g / L.

[0035] The construction process of the engineered strain is described in detail below.

[0036] MccJ25 gene cluster

[0...

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Abstract

This solution provides an engineering strain that highly expresses MccJ25 to solve the problem of low expression of Microcin J25 in the prior art. Constructed by the following method: respectively carry out gene manipulation on mcjABCD to become one direction, and share a promoter T7 in Escherichia coli, clone it in the vector pBR322, and realize high-efficiency expression in Escherichia coli, the expression amount is 2.4g / L. In this project, a new expression vector was constructed, and the expression vector of Escherichia coli and Lactobacillus plantarum were respectively constructed to efficiently express MccJ25. At the same time, the directed evolution technology was used to carry out molecular evolution of the MccJ25 gene cluster to realize the high-efficiency expression of MccJ25. The extracellular expression in Escherichia coli and the intracellular expression in Lactobacillus plantarum reached 4.1g / L and 2.3g / L respectively, reaching the level of industrialization and laying the foundation for the application of biological veterinary drugs and feed additives in the next step .

Description

technical field [0001] The solution belongs to the technical field of genetic engineering and the technical field of fermentation optimization, and specifically relates to an engineered strain that expresses MccJ25 with high efficiency and a fermentation process thereof. Background technique [0002] The lariat peptide MccJ25 is a bacteriocin-like antimicrobial peptide first discovered in Escherichia coli AY25, isolated from human feces (Blond et al., 1999). The synthesis of the lasso peptide MccJ25 involves four genes, mcjA, mcjB, mcjC and mcjD (Solbiati et al., 1996). [0003] The mcjA gene is 174 bp long and is the precursor of MccJ25. During the process of maturation, the N-terminal 37aa is excised to form MccJ25. Lasso peptide Mcc J25, wherein the 8 amino acids at the N-terminal form a circular ring structure, and the 9-21 amino acids form a β hairpin structure, which runs through the ring. Through biochemical and nuclear magnetic resonance studies, it was found that ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/74C12N15/31C12R1/19C12R1/25
CPCC07K14/245C12N15/70C12N15/746
Inventor 赵珊孟凡杰杨贤彬
Owner 安杰利(重庆)生物科技有限公司
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