Genetically modified violacein biosynthetic gene cluster, recombinant expression vector, engineering bacterium and application thereof

A purple bacteriocin and genetically engineered bacteria technology, applied in the biological field, can solve the problems such as the inability to meet the strict requirements of cost, and achieve the effects of low price, simple medium components, and increased yield

Active Publication Date: 2020-10-09
XINXIANG MEDICAL UNIV
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  • Claims
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Problems solved by technology

Although the current fermentation yield of genetically engineered bacteria has been greatly i

Method used

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  • Genetically modified violacein biosynthetic gene cluster, recombinant expression vector, engineering bacterium and application thereof
  • Genetically modified violacein biosynthetic gene cluster, recombinant expression vector, engineering bacterium and application thereof
  • Genetically modified violacein biosynthetic gene cluster, recombinant expression vector, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0045] Example 1: Obtainment of wild-type violacein biosynthesis gene cluster (vioABCDE)

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

Example Embodiment

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

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

Example Embodiment

[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, using the primer pair vioB-RBSm-PF / PR to use reverse PCR amplification technology to establish the ribosome binding site of the vioB gene in the purple bacteriocin biosynthesis gene cluster Introduce site-directed mutations. The PCR product was first incubated with DpnI enzyme at 37°C for 1 hour to eliminate the PCR template, and then the enzymatic digestion product was transformed into E.coli DH5α chemically competent cells and coated with Amp resistant LB plates.

[0051] (2) The plate is placed in an incubator and incubated at 37°C overnight to produce new transformants, and 3 transformants are picked in parallel for DNA sequencing verification.

[0052] (3) For transformants with correct sequencing, the corresponding mutant plasmid Vio12472-vioB-RBSm i...

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Abstract

The invention discloses a genetically modified violacein biosynthetic gene cluster, a recombinant expression vector, engineering bacteria and application thereof. The ribosome binding site of the violacein biosynthetic gene cluster is subjected to site-specific mutagenesis and codon deletion mutation transformation, so that effective translation of violacein synthetic protein is promoted, and thetransformed gene cluster with remarkably improved production potential is obtained; by introducing the modified gene cluster or the recombinant vector containing the modified gene cluster into host bacteria, engineering bacteria for high yield of violacein are obtained; furthermore, fermentation conditions of engineering bacteria are optimized, and a method for separating and purifying violacein is provided. The genetic engineering strain constructed by the invention has the advantages that the yield of violacein can be obviously improved; different from conventional low-temperature fermentation of violacein at 20 DEG C or 25 DEG C, the violacein metabolite can be efficiently produced under the temperature condition of 25-37 DEG C, the problem of high cooling cost in the fermentation process is solved, and the invention is suitable for large-scale production.

Description

technical field [0001] The invention relates to a violacein biosynthetic gene cluster through genetic modification, a recombinant expression vector, an engineering bacterium and an application 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 oxidative condensation of two tryptophan molecules. 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-purple characteristics. Therefore, violacein has potential application value in a wide range of industrial markets such as medicine, health care, cosmetics, food additives, insecticides, textiles, and toys. [0003] Due to the good application potential of violacein compounds, a lot of work has been done to improve their production in fermentation broth. However, due to the low productivity and poten...

Claims

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Application Information

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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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