Deletion of the gene and its application, the strain of the gene deletion and its application in improving the production of microbial secondary metabolites

A gene and strain technology, applied in microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of low yield of deoxyvioletine, less research on properties and biological activities, and difficult separation.

Active Publication Date: 2022-02-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of deoxyviolet violacein is very low, and its separation is difficult, so there are few studies on its properties and biological activities.

Method used

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  • Deletion of the gene and its application, the strain of the gene deletion and its application in improving the production of microbial secondary metabolites
  • Deletion of the gene and its application, the strain of the gene deletion and its application in improving the production of microbial secondary metabolites
  • Deletion of the gene and its application, the strain of the gene deletion and its application in improving the production of microbial secondary metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Method for continuous evolution of haploid Saccharomyces cerevisiae phenotype

[0044] The invention provides a research method for accelerating the phenotypic evolution of strains by utilizing synthetic Saccharomyces cerevisiae circular chromosomes, using PDV, a downstream metabolite of aromatic amino acids as a marker, to generate a large number of phenotypic variant yeast strains by inducing circular chromosome rearrangement.

[0045] 1. Design primers PCR-amplify the open reading frames (ORFs) of vioA, vioB and vioE from the BioBrick element library, and recover DNA fragments from agarose gel.

[0046] 2. Designing primers The promoter and terminator were amplified from Saccharomyces cerevisiae BY4741 genomic DNA, wherein there was a 40 base pair (bp) overlapping region between the promoter, the open reading frame and the terminator. DNA fragments were recovered from agarose gel.

[0047] 3. Digest the pRS416 plasmid with EcoRI restriction enzyme and perf...

Embodiment 2

[0068] Example 2 The yield of Saccharomyces cerevisiae purple bacteriocin precursor metabolizing strains increased continuously

[0069] Circular synthetic yeast chromosomal rearrangements can achieve continuous improvement in the phenotype of yeast strains. We performed 5 rounds of rearrangement experiments on the strain (yWJS067), expecting to obtain a strain with improved PDV production. For the final selected strains, the percentages of dark colonies were 7.79%, 3.76%, 4.09%, 3.88%, 3.84% and 1.07%, respectively. For each round of rearrangement experiments, the strain with the darkest visible color and similar growth fitness compared to the parental strain was selected for quantification of PDV productivity by HPLC. After six rounds of rearrangement experiments, we obtained a strain with approximately seven-fold increased PDV production, and this strain had a favorable growth phenotype.

[0070] Table 4 figure 2 araw data

[0071]

[0072] table 5 figure 2 braw d...

Embodiment 3

[0075] Example 3 Continuous variation of chromosome number of Saccharomyces cerevisiae

[0076] In order to shed light on the genetic variation generated in ring_synV, the applicant performed whole genome sequencing (WGS) analysis on some strains induced to rearrangement (SCRaMbLEants), yWJS044, yWJS047, yWJS067, yWJS184 and yWJS321. Among the rearranged strains analyzed, yWJS044, yWJS047 and yWJS067 were obtained from the starting strain yWJS1 after the first round of SCRaMbLE, yWJS184 was obtained from yWJS067 after the third round of SCRaMbLE, and yWJS321 was obtained from yWJS184 , The rearranged strain obtained after the fifth round of SCRaMbLE.

[0077] image 3The boxplot based on WGS results in Zhongwei shows the coverage depth of different chromosomes, representing the copy number of different chromosomes, and shows that the haploid Saccharomyces cerevisiae synthetic circular chromosome V SCRaMbLE can lead to chromosomal aneuploidy. Strain yWJS044 (aneuploid chromos...

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Abstract

The invention relates to the field of synthetic biology, in particular to gene deletion and its application, the gene deletion strain and its application in increasing the yield of microbial secondary metabolites. It is found through experiments that the deletion of genes YEL017C‑A, YEL017W and / or YER182W can increase the production of deoxyviobacterin precursor, deoxyviobacterin, violacein precursor and / or violacein.

Description

technical field [0001] The invention relates to the field of synthetic biology, in particular to gene deletion and application thereof, strains lacking the gene and application in improving the yield of microbial secondary metabolites. Background technique [0002] Violacein and deoxyviolacein are metabolites of microorganisms, belonging to indole derivatives, which are formed by the oxidative condensation of two tryptophan molecules. Since the discovery of purple bacteriocin in the late 19th century, people have carried out a lot of exploration on its biological function, and found that purple bacterin has strong biological activities: (1) with broad-spectrum antibacterial activity; (2) anti-oxidation; (3) ) Anti-tumor cells; (4) Anti-virus; (5) Protozoa; (6) Process various fabrics as dyes. In conclusion, violetobactin has high medical value and industrial application prospect. [0003] Among the violacein-producing strains that have been found, C. violaceum is the most ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P17/16C12R1/865
CPCC12P17/165C07K14/395
Inventor 元英进谢泽雄王娟李炳志
Owner TIANJIN UNIV
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