Screening And Heterologous Expression Of Aromatic Polyketide Biosynthesis Gene Clusters From Metagenomic Library

The aboundant microbial resources lead to the diversity of biological active microorganism metabolites.Some famous antibiotics had been found from cultured environmental microorganisms,such as tetracycline,erythromycin,and rifampicin.However,the traditional method of cultivation and isolation of microorganisms has lead to high repetition rate up to 99%of known compounds,and its more differcult to obtain new structures of the compounds.Studies have shown that less than 1%of the microorganism can be cultured by the conventional laboratory culture conditions.The vast majority of microorganisms can not be cultured by conventional culture methods.Metagenomics technology,did not dependent on the microbial culture technology.By metagenomics technology,the microbial DNA was extracted directly from environmental samples.Then the repaired DNA was connected to suitable vector and transformed into host bacteria,to establish the metagenomic library.Then the libraries were analyzed and screened to obtain new genes.This technology broke through the bottleneck of the culture-based method,being a new method with great potential.We have constructed a soil metagenomic library which was collected from Everest.We have cloned the type ? polyketide synthase genes from our soil metagenomic library.A pair of degenerate primers was designed according to the type ? polyketide homologous gene KSa,and the aromatic polytetide gene was screened by the degenerate primers.Then the positive clones was screened again by designed specific primers.26 different clones that contained the type ? polyketide homologous gene KS? were obtained.Further sequencing was carried out to determine which one contained complete gene cluster and findings of overlapping clones was also prefermed.The sequencing result of No.493 revealed that it enconding a complete aromatic polytetide biosynthetic gene cluster of 39896 bp.No.493 positive clone was expressed in Streptomyces albus J1074 and new peak was detected in its fermentation extraction.At the same time,the fermentation extracts showed antibacterial activity.