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Genetically modified purple bacteriocin biosynthesis gene cluster, recombinant expression vector, engineered bacteria and their applications

A technology of purple bacteriocin and genetically engineered bacteria, applied in the biological field, can solve problems such as the inability to meet strict cost requirements, and achieve the effects of improving production potential, efficient production and low price

Active Publication Date: 2022-07-22
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current fermentation yield of genetically engineered bacteria has been greatly improved, it still cannot meet the strict requirements for cost in future industrial production

Method used

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  • Genetically modified purple bacteriocin biosynthesis gene cluster, recombinant expression vector, engineered bacteria and their applications
  • Genetically modified purple bacteriocin biosynthesis gene cluster, recombinant expression vector, engineered bacteria and their applications
  • Genetically modified purple bacteriocin biosynthesis gene cluster, recombinant expression vector, engineered bacteria and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Acquisition of wild-type violet bacteriocin biosynthesis gene cluster (vioABCDE)

[0046] The genomic DNA of Chromobacterium violaceum (ATCC 12472) was isolated by the rapid bacterial genomic DNA extraction kit from Shanghai Sangon Biotechnology Co., Ltd., and the PCR amplification template of the violacein gene cluster clone was obtained; then the primer pair Vio-pETduet-PF / PR, via Takara High-Fidelity Enzyme Prime GXL DNA polymerase amplified vioABCDE (SEQ ID NO: 5) of 7325 bp.

Embodiment 2

[0047] Example 2: Construction of recombinant expression vector Vio12472

[0048] Using the recombinant cloning kit of Nanjing Vazyme Company, the vioABCDE operon (7325bp) was recombined into the KpnI site of the pETduet-1 expression vector. The plasmid formed after sequencing verification was named Vio12472 (such as figure 1 ).

Embodiment 3

[0049] Example 3: Construction of recombinant expression vectors Vio12472-vioB-RBSm, Vio12472-vioC-RBSm, Vio12472-vioD-RBSm, Vio12472-vioE-RBSm

[0050] (1) Using the wild-type plasmid Vio12472 obtained in Example 2 as a template, the primer pair vioB-RBSm-PF / PR was used to amplify the ribosome site of the vioB gene in the violetin biosynthesis gene cluster by inverse PCR amplification technology. Introduction of site-directed mutagenesis. The PCR product was first incubated with DpnI enzyme at 37°C for 1 h to eliminate the PCR template, and then the enzyme digested product was transformed into E. coliDH5α chemically competent cells and coated with Amp-resistant LB plates.

[0051] (2) The plate was placed in an incubator, and new transformants were generated after overnight incubation at 37°C, and three transformants were picked in parallel for DNA sequencing verification.

[0052] (3) For the transformants with correct sequencing, a conventional plasmid extraction method wa...

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Abstract

The invention discloses genetically modified violetin biosynthesis gene clusters, recombinant expression vectors, engineering bacteria and applications thereof. The ribosome binding sites of violacein biosynthesis gene clusters are modified by site-directed mutation and codon deletion mutation , to promote the efficient translation of the violetin synthesis protein, and obtain a modified gene cluster with significantly improved production potential; by introducing the above-mentioned modified gene cluster or a recombinant vector containing the modified gene cluster into the host bacteria, an engineered bacteria with high production of violetobactin was obtained; The fermentation conditions of the engineered bacteria were further optimized and a method for the separation and purification of violetobactin was provided. The genetically engineered strain constructed by the present invention can not only significantly improve the yield of violetobactin, but also can efficiently produce violetobactin metabolism under the temperature condition of 25-37 ℃, which is different from the conventional low-temperature fermentation of violetobactin at 20°C or 25°C. The product solves the problem of high cooling cost in the fermentation process, and is suitable for large-scale production.

Description

technical field [0001] The invention relates to a genetically modified purple bacteriocin biosynthesis gene cluster, a recombinant expression vector, an engineered bacteria and applications thereof, and belongs to the field of biotechnology. Background technique [0002] Violacein is a metabolite produced by microorganisms. It belongs to indole derivatives. It is formed by the oxidative condensation of two tryptophan molecules. It has anti-tumor, anti-virus, anti-Staphylococcus aureus infection, anti-oxidation, It has good biological activities such as anti-malarial and immune regulation. At the same time, it can be used as a natural pigment due to its own blue-violet characteristics. Therefore, violetobactin has potential application value in a wide range of industrial markets such as medicine, health care, cosmetics, food additives, pesticides, textiles, toys, etc. [0003] Due to the good application potential of violetobactin compounds, a lot of work has been done to im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/70C12N15/65C12N1/21C12P17/16C07D403/14C12R1/19
CPCC12N9/00C12N15/70C12N15/65C12P17/165C07D403/14
Inventor 张玉阳陈红萍牛志远杨荣迪李志坤李艳娇
Owner XINXIANG MEDICAL UNIV
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