Genetically engineered high-producing strain Streptomyces diastatochromogenes and method for increasing yield of epsilon-polylysine

A technology of Streptomyces chromogenes and high-yielding strains is applied in the biological field, which can solve the problems of increasing ε-polylysine and the like, and achieve the effect of improving the fermentation level

Active Publication Date: 2020-07-31
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on improving the production of ε-polylysine by genetic engineering, especially by overexpressing the acetyl-CoA acetyltransferase (acetyl-CoAacetyltransferase) gene catF

Method used

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  • Genetically engineered high-producing strain Streptomyces diastatochromogenes and method for increasing yield of epsilon-polylysine
  • Genetically engineered high-producing strain Streptomyces diastatochromogenes and method for increasing yield of epsilon-polylysine
  • Genetically engineered high-producing strain Streptomyces diastatochromogenes and method for increasing yield of epsilon-polylysine

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Experimental program
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Effect test

Embodiment 1

[0070] Obtaining the target catF gene: design the primer sequence according to the catF gene, introduce EcoRI and XbaI enzyme cutting sites at both ends of the gene catF, and add 6 nucleotides to the upstream 5' end of the catF nucleotide sequence to form a restriction The site of the sex endonuclease XbaI, add 6 nucleotides at the downstream 5' end of the catF nucleotide sequence to form the site of the restriction endonuclease EcoRI to extract the amylase Streptomyces chromogenes TUST genomic DNA As a template, use the sequence table SEQ ID No.2 and SEQ ID No.3 as the upstream and downstream primers respectively to carry out PCR reaction to amplify the catF gene in the amylase Streptomyces chromogenes TUST, the full length is 1158bp, see the sequence table SEQ ID No .1.

[0071] PCR reaction system: 2×phanta max buffer 25 μL, dNTP mixture (10mM) 1 μL, template (20ng / ul) 1 μL, upstream and downstream primers ((10 μM)) 2 μL each, DMSO 2 μL, phanta max×Super-Fidelity DNA Polyme...

Embodiment 2

[0073] Construction of recombinant plasmid pIMEP-catF containing catF gene:

[0074] The catF gene fragment amplified by PCR was double-digested with XbaI and EcoRI, and the erythromycin promoter PermE* plasmid pIMEP was double-digested with XbaI and EcorI, and then the catF gene fragment was connected to the double-digested pIMEP plasmid. The corresponding XbaI and EcoRI restriction sites were used to obtain the recombinant plasmid pIMEP-catF of the ligation product. Such as figure 1 shown.

Embodiment 3

[0076] Transformation of recombinant plasmid pIMEP-catF:

[0077] Take the recombinant plasmid pIMEP-catF of the ligation product and add it to the centrifuge tube containing Escherichia coli JM109 competent cells that have been melted in the ice bath, flick the tube wall, mix well, and put it in the ice bath for 30 minutes. Heat shock at 42°C for 90s, then immediately ice bath for 5min (do not move during this process). Under sterile conditions, add 900 μL of LB medium to the centrifuge tube, mix by pipetting, and incubate with shaking at 37°C and 200 r / min for 45 minutes. Centrifuge the centrifuge tube at 12000r / min for 1min, remove 900μL of supernatant, blow and mix the remaining liquid with a pipette, and spread it on the LB solid plate containing apramycin resistance. Incubate the LB plate upside down at 37°C overnight until a single colony is clearly identifiable, pick positive transformants for colony PCR verification, and the results are as follows figure 2 As shown...

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Abstract

The invention relates to a genetically engineered strain Streptomyces diastatochromogenes CATF with high yield of epsilon-polylysine, and a method for improving the fermentation level of the epsilon-polylysine. A construction method of the high-producing gene genetically engineered strain comprises the following steps: step 1, constructing a plasmid for expressing acetyl-CoA acetyltransferase CATFgene, wherein a gene CATF sequence fragment is controlled by an erythromycin promoter erm* on the pIMEP plasmid; and step 2, acquiring a strain expressing the catF gene, namely the genetically engineered strain Streptomyces diastatochromogenes CATF with high yield of the epsilon-polylysine. Experiments prove that the epsilon-polylysine production capacity of the genetically engineered streptomyces strain is remarkably improved by 27.91% compared with that of an original strain Streptomyces diastatochromogenes TUST under the same condition, and an excellent strain is provided for epsilon-polylysine production.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a gene engineering high-yield amylase streptomyces chromogenic strain CATF and a method for increasing the yield of ε-polylysine. Background technique [0002] ε-polylysine is one of the two natural amino acid homopolymers discovered so far (the other is γ-polyglutamic acid). After the discovery of the first strain of ε-polylysine-producing bacteria, ε-polylysine was obtained through soil screening. - Polylysine-producing bacteria all belong to the genera Streptomyces, Streptoverticillum, Kitasatospora and Cinnamomum . The distribution of ε-polylysine producing bacteria was mainly restricted to the filamentous bacteria Streptomycetes and ergot fungi. ε-polylysine has a broad antibacterial spectrum, and has inhibitory effects on Gram-positive bacteria, Gram-negative bacteria, fungi and some viruses. It has good thermal stability and can be directly added to food for process...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12P13/02C12R1/525
CPCC12N9/1029C12Y203/01009C12N15/76C12P13/02
Inventor 谭之磊李小娜贾士儒董天宇唐昆鹏崔建东王贺莉魏希庆
Owner TIANJIN UNIV OF SCI & TECH
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