Cloning And Analysis Of Type?Polyketide Synthase Genes In Metagenomic Library

Microorganisms are the important source of bioactive natural substances.Many drugs in clinicals are natural products and their derivatives such as cephalosporin,lovastatin and rapamycin.Soil microbes play a crucial role in the biogeochemical cycle and sustainable development.According to the studies,the number of bacterial species in the world ranges from 107 to 109.It greatly increases the rate of studies and utilization of microbes to produce the natural bioactive products.However,only 1%of the environmental microorganisms can be cultured in the laboratory which greatly limits the use of microbial resources.Extarcting environmental DNA to construct the metagenomic library and analysis the gene clusters of natural products make it expanded greatly of the space in utilization of microbial genetic resources.There are great chances to get new natural bioactive products.This is metagenomics.In order to explore and analysis the potential abilities of microorganisms to produce natural products and clone related genes of polyketide synthases,we have constructed the soil metagenomic library and analyzed the related genes of type ? polyketide synthases.Polyketides are an important part of natural products.Polyketides are synthesized by polyketide synthases and type ? polyketide synthase is modular.Each module can be divided into different domains.Among the natural products and their derivatives,more than one-third is polyketides.We constructed the soil metagenomic library of environmental DNA which is collected from Yunnan soil.We had cloned the type ? polyketide synthase genes of environmental microbes through metagenomics.We have got following results.High quality environmental DNA was directed extracted from Yunnan soil to construct the soil metagenomic library of cosmid.By calculating,the total number of clones in Yunnan soil metagenomic library is 107 and the average size of the insert DNA is 37kb to 38kb.The large number of clones has ensured that the cosmid library covers the DNA of almost soil microbes in theory.A pair of degenerate primers named KS is designed according the conserved domains of genes of keto-synthases among type ? polyketide synthases.We screened PKS genes in the soil metagenomic library according the KS degenerate primers primitively.Then the soil library was screened again by specific primers designed according sequences of the primary PCR products of degenerate primers.We get 67 different clones that contained KS domain of type ? polyketide synthases depending on homologous screening.After preliminary analysis of the sequences,we find a sequence encoding PKS/NRPS.It is 12952bp and it contains two open reading frames.In conclusion,a soil metagemonic library of Yunnan was constructed in the research.67 clones was screened by KS degenerate domains depending on the homologous screening and one of them contains genes encoding PKS/NRPS.The feasibility of the soil metagenomics to screening new polyketide synthases has been fully verified.The research lays the solid foundation for further study of new type ? polyketide synthases.Additionally,a new way of exploring microbial resources,discovering of new drugs and its genes was established.