Carbon source absorption expression system, recombinant bacteria and application

A technology of absorption system and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of affecting the yield and fermentation titer of the target product, affecting the industrial application, etc.

Active Publication Date: 2020-09-01
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This problem seriously affects the yield of the target product and the unit fermentation titer, further affecting the industrial application

Method used

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  • Carbon source absorption expression system, recombinant bacteria and application
  • Carbon source absorption expression system, recombinant bacteria and application
  • Carbon source absorption expression system, recombinant bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Construction of carbon source absorption system.

[0027] The carbon source absorption system described in this example is composed of a promoter and a gene for absorbing carbon source connected by a restriction endonuclease, the promoter is promoter P2 or promoter P4, and the gene for absorbing carbon source is Gene TP2 or TP5.

[0028] Using the genome of Streptomyces icebergi BC04 as a template, primers P2-F / P2-R were used to amplify the promoter P2(P sbi_07857 ) sequence, as shown in SEQ ID No.3; Utilize primer P4-F / P4-R to amplify promoter P4 (P sbi_06929 ) sequence, as shown in SEQ ID No.4.

[0029] Using the genome of Streptomyces iceberg BC04 as a template, using primers TP2-F / TP2-R to amplify the gene TP2 that absorbs carbon sources, the TP2 gene contains 3 gene fragments, and the registration of the 3 gene fragments on GenBank The numbers are GeneID: 11614576 (encoding transmembrane protein), 11614577 (encoding transmembrane protein) and 11614578...

Embodiment 2

[0031] Example 2. Construction of a recombinant vector containing the carbon source uptake system described in Example 1.

[0032] Using the genome of Streptomyces icebergi BC04 as a template, primers P1-F / P1-R were used to amplify the promoter P1(P sbi_05102 ) sequence, the P1 promoter and the TP2 gene obtained in Example 1 and EcoRI, XbaI digested linearized pSET152 plasmid backbone using a multi-fragment one-step cloning kit (purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.) through Gibson Assembling method, the plasmid P1-TP2 of the promoter P1 controlling the sugar absorption system was obtained, and the plasmid P1-TP5 of the promoter P1 controlling the sugar absorption system was obtained by referring to the same method.

[0033] The promoter P2 and the plasmid P1-TP2 were respectively digested by NotI and SpeI and ligated to obtain the recombinant vector P2-TP2 of the sugar absorption system controlled by the promoter P2.

[0034] The promoter P4 and the plasmi...

Embodiment 3

[0037] Example 3. Construction of recombinant bacteria that synthesize Streptomyces secondary metabolites.

[0038] The recombinant vectors P2-TP2, P4-TP2, P2-TP5, and P4-TP5 prepared in Example 2 were introduced into Streptomyces glaciferi BC04 by conjugative transfer, respectively, to obtain recombinant strains for the synthesis of milbemycin BC04TP2P2 and BC04TP2P4 and BC04TP5P2 and BC04TP5P4.

[0039] The recombinant vectors P2-TP2 prepared in Example 2, P4-TP2, P2-TP5 and P4-TP5 were introduced into Streptomyces avermitilis NEAU1069 by conjugative transfer respectively to obtain recombinant strains NEAUTP2P2 for synthesizing abamectin, respectively. NEAUTP2P4, and NEAUTP5P2 and NEAUTP5P4.

[0040]The recombinant vectors P2-TP2, P4-TP2, P2-TP5 and P4-TP5 prepared in Example 2 were introduced into Streptomyces coeruleus NMWT1 by conjugative transfer, respectively, to obtain recombinant strains ScyTP2P2 for the synthesis of nimoctine , ScyTP2P4, ScyTP5P2 and ScyTP5P4.

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Abstract

A carbon source absorption expression system, recombinant bacteria and application belong to the technical field of gene engineering. In order to increase the yield of streptomyces secondary metabolites, the invention provides a carbon source absorption system. The carbon source absorption system contains a promoter and a gene for absorbing a carbon source, wherein the nucleotide sequence of the promoter is shown as SEQ ID NO.3 or SEQ ID NO.4, and the nucleotide sequence of the gene for absorbing the carbon source is shown as SEQ ID NO.1 or SEQ ID NO.2. By converting the carbon source absorption system into streptomyces to obtain an engineering strain for fermentation, the sugar absorption efficiency can be effectively improved, the sugar residue is reduced, and the output and yield of a target product are improved; fine adjustment expression of the carbon source absorption system enables the carbon source absorption system to be matched with metabolic behaviors of a host, which is very important for increasing the yield of secondary metabolites in streptomyces.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a carbon source absorption expression system, recombinant bacteria and applications. Background technique [0002] In the industrial fermentation process of Streptomyces secondary metabolites, the low utilization rate of carbon source substrates by strains is a common problem. This problem has seriously affected the yield of the target product and the unit fermentation titer, and further affected the industrial application. The biosynthesis of Streptomyces secondary metabolites was produced in a large amount during the stable growth period of the strain, and the primary metabolism of the strain was almost stagnant at this stage. In particular, the uptake rate of Streptomyces on carbon source substrates (mainly soluble sugar) will drop sharply after the strain enters the stationary phase. Carbon source is an important nutrient for the synthesis of target s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/76C12P19/62C12P17/18C12R1/465
CPCC07K14/36C12N15/76C12P17/181C12P19/623
Inventor 向文胜李珊珊金品娇张艳艳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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