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
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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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