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Gene cluster expression strengthened recombinant streptomyces diastatochromogenes and construction method thereof

A technology for producing Streptomyces chromosum and amylase, which is applied in the field of genetic engineering, can solve the problems to be improved, increase the metabolic flux, involve complicated problems and the like, and achieve the effect of increasing the yield of toyomycin

Inactive Publication Date: 2015-01-28
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the previous study, our research group used the toyocamycin-producing strain Streptomyces chromogenes (Streptomyces chromogenes) with independent intellectual property rights Streptomyces diastatochromogenes ) 1628 as the research object, cloned some structural genes in the gene cluster, such as toyB, toyF, etc., and tried to S. diastatochromogenes overexpressed in 1628 and also cloned S. diastatochromogenes The pleiotropic regulatory factor adpA responsible for secondary metabolism in 1628, but the increase in the yield of toyocamycin is only 20%-30%, which still needs to be improved for industrial production
Using metabolic engineering technology to increase the copy number of the original strain biosynthetic toyocamycin gene cluster instead of a single structural gene, thereby increasing the metabolic flux of toyocamycin synthesis and increasing the production of toyocamycin, because the problems involved are complex and there is no correlation to report

Method used

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  • Gene cluster expression strengthened recombinant streptomyces diastatochromogenes and construction method thereof
  • Gene cluster expression strengthened recombinant streptomyces diastatochromogenes and construction method thereof
  • Gene cluster expression strengthened recombinant streptomyces diastatochromogenes and construction method thereof

Examples

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Effect test

Embodiment 1

[0029] Example 1: Amplification and biological function verification of gene cluster toyAM

[0030] To S. diastatochromogenes 1628 chromosome genome as template, using Ptoy F Bam HI and PtoyF R Bam HI is the primer, PCR amplification obtained contains Bam The gene cluster toy AM gene at the HI restriction site was connected with pMD18-T Vector to construct the cloning vector pMD18-T-toy AM. The cloning vector pMD18-T-toy AM was transformed into the recipient E. coli and spread on After cultured overnight at 37°C on an ampicillin-resistant LB agar plate, the positive transformants were randomly selected and identified by restriction enzyme digestion and sent to Shanghai Shenggong for sequencing and sequence analysis. Bam HI digestion to get both ends containing Bam The gene cluster toy AM at the HI restriction site, and the same use Bam The HI digested and dephosphorylated Streptomyces integrative shuttle expression vector pSET152 was ligated to obtain the recombinant shutt...

Embodiment 2

[0036] Example 2: Construction of Recombinant Strain of Streptomyces chromogenes

[0037] The recombinant plasmid pSET152-toy AM was integrated into the amylase Streptomyces chromogenes ( Streptomyces diastatochromogenes ) On the chromosome of 1628, a few single colonies were randomly picked on the apramycin-resistant plate and cultured in CP medium for many times, and then the chromosomes were extracted. PCR experiments can amplify the apramycin resistance gene apr ( Figure 5 ), proving that the recombinant amylase Streptomyces chromogenes 1628-TOYAM was successfully constructed and genetically stable.

Embodiment 3

[0038] Example 3: Verification of fermentation performance of amylase-producing Streptomyces chromogenes original and recombinant bacteria

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Abstract

The invention discloses a gene cluster expression strengthened recombinant streptomyces diastatochromogenes and a construction method thereof. The gene cluster expression strengthened recombinant streptomyces diastatochromogenes excessively expresses a whole toy gene cluster for biosynthesizing toyocamycin and has stronger toyocamycin synthetic capability than streptomyces diastatochromogenes 1628. A construction process comprises the following steps: 1) constructing an expression vector pSET152-toyAM; and 2) integrating the expression vector into chromosome of the streptomyces diastatochromogenes by utilizing a conjugative transferring method to obtain an engineering bacterium. According to the construction method disclosed by the invention, specificity of the vector pSET152-toyAM is integrated into the chromosome of the streptomyces diastatochromogenes 1628 by utilizing the conjugative transferring method to obtain an engineering bacterium with genetic stability. Compared with an original strain, the gene cluster expression strengthened recombinant streptomyces diastatochromogenes has the advantage that the yield of toyocamycin is increased by 4.5 times.

Description

technical field [0001] The invention relates to improving the yield of toyocamycin by strengthening the expression of gene cluster toy AM in amylase chromogenic streptomyces, 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12P19/40C12R1/525
CPCC12N15/52
Inventor 马正俞晓平徐显皓陶立彬申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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