ToyG expression-enhanced recombined streptomyces diastatochromogenes and construction method and uses thereof

A technology for producing Streptomyces chromogenes and amylase, which is applied in the field of genetic engineering to achieve the effect of increasing yield

Active Publication Date: 2014-05-28
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using metabolic engineering technology to further improve the yield of toyocamycin by increasing the metabolic flux of toyocamycin has not been reported because of complex issues such as the establishment of an expression system.

Method used

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  • ToyG expression-enhanced recombined streptomyces diastatochromogenes and construction method and uses thereof
  • ToyG expression-enhanced recombined streptomyces diastatochromogenes and construction method and uses thereof
  • ToyG expression-enhanced recombined streptomyces diastatochromogenes and construction method and uses thereof

Examples

Experimental program
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Embodiment 1

[0031] Example 1: Amplification of target gene and construction of recombinant amylase-producing Streptomyces chromosome

[0032] To S. diastatochromogenes 1628 chromosome genome as template, using PtoyG F Nde I and PtoyG R Not I is the primer, PCR amplification obtained contains Nde I and Not I The toyG genes at the two restriction sites are connected with pMD18-T Vector to construct the cloning vector pMD18-T-toyG ( Nde I + Not I), the cloning vector pMD18-T-toyG ( Nde I + Not I) Transform into recipient Escherichia coli, spread it on an ampicillin-resistant LB agar plate, culture overnight at 37°C, randomly select positive transformants for identification and send them to Shanghai Shenggong for sequencing and sequence analysis. Nde I and Not I double digestion to get Nde I and Not I. The toyG genes at the two restriction sites are connected with the same double-enzyme digested Streptomyces integrative shuttle expression vector pIB139 to obtain the recombin...

Embodiment 2

[0038] Example 2: Verification of fermentation performance of amylase-producing Streptomyces chromogenes original and recombinant bacteria

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Abstract

The invention discloses a toyG expression-enhanced recombined streptomyces diastatochromogenes and a construction method and uses thereof. The recombined streptomyces diastatochromogenes excessively expresses that the key enzyme of biosynthetic toyocamycin-an adenosine succinic acid synthetase encoding gene toyG has a toyocamycin synthesis capacity higher than streptomyces diastatochromogenes 1628. The construction process is as follows: (1) constructing an expression vector pIB139-toyG; (2) utilizing a conjugational transfer method to integrate the expression vector into a chromosome of streptomyces diastatochromogenes to obtain engineering bacteria. A promoter permE on the vector pIB139 is utilized to open the expression of the toyG gene, and the conjugational transfer method is utilized to integrate the specificity of the vector pIB139-toyG into the chromosome of streptomyces diastatochromogenes 1628 to obtain the genetic-stability engineering bacteria. Compared with parent strains, the enzyme activity of recombinant strain adenosine succinic acid synthetase is increased by at least 1.2 times, and the yield of toyocamycin is increased by at least 27.1 percent.

Description

technical field [0001] The invention relates to improving the yield of toyocamycin by strengthening the expression of the toyG gene in the amylase streptomyces chromogenes, and belongs to the technical field of genetic engineering. Background technique [0002] Toyocamycin is a new type of nucleoside antibiotics with the molecular formula C 12 h 13 N 5 o 4 , Ribose C 1 The deazapurine ring similar to guanine is connected, and the core structure is a pyrropyrimidine nucleoside analogue. The mechanism of action is mainly to affect the growth of bacteria by inhibiting the transcription of microorganisms, and the research reports on its biological activity are mainly concentrated in the field of clinical medicine. Recent studies have found that toyocamycin has a good control effect on a variety of plant diseases, long-term use will not cause environmental pollution, and it also has a certain regulatory effect on plant growth. Therefore, toyocamycin has great application po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N1/21C12N15/76C12P19/40C12R1/525
Inventor 马正俞晓平陶立彬申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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