Screening And Heterologous Expression Of Biosynthetic Gene Clusters Containing Halogenase And Aromatic Polyketide Synthase From Metagenomic Library

Microbial natural products are important sources of drugs.At present,many medicines have been developed based on microbial natural products,which are widely used in the clinic,agriculture and animal husbandry,such as tetracycline and vancomycin.Soil is a huge microbial resource,and it is estimated that there are as many as 105 species of microorganisms in 1 g soil.For the study of microbial natural products,the traditional culture-based method has been used for a long time.But only 1%of microorganisms can be isolated and cultured.Therefore,the numbers of microorganisms that can be investigated is greatly limited.The method of metagenomes is to study the entire DNA of microorganisms in a specific environment,which avoided the isolation and culture of microorganisms in the traditional culture-based method and enlarged the number of the studied microorganisms.In this study,sequence-driven screening method was used to screen the soil metagenomic library,in order to obtain novel biosynthetic gene clusters of microbial natural products.Most halometabolites and aromatic polyketide compounds have good antibacterial and anti-tumor activities,such as chloramphenicol and tetracycline,which are widely used in the pharmaceutical industry.But with the emergence of new diseases and pathogens with drug resistance,the demand for novel active compounds is more and more urgent.So it is very significant to carry out the experiment for halometabolites and aromatic polyketide compounds with unique structure and novel activities.The Yunnan No.3 metagenomic library of soil has been screened by halogenase degenerate primer and aromatic polyketide synthase degenerate primer,and the monoclones with halogenase gene and aromatic polyketide synthase gene are obtained respectively.For the monoclones with halogenase gene,the exogenous environmental DNA gene is integrated into the genome of the host Streptomyces albus by conjugal transfer,and then the fermentation of S.albus with exogenous DNA gene is carried out by using different media.After fermentation,the fermentation products are extracted and analyzed by HPLC and TLC in order to obtain the new type of natural products.At the same time,screen for overlapping clones for a number of monoclones with halogenase gene by the use of terminal gene sequences.Finally,seventy monoclones with halogenase gene and nine monoclones with aromatic polyketide synthase gene are obtained in the experiment.For sixty monoclones with halogenase gene,the exogenous environmental DNA gene is integrated into the genome of S.albus by conjugal transfer successfully and the fermentation of all the S.albus were carried out.However,no new natural products have been found when the fermentation products were detected.At the same time,5 overlapping clones of NO.718 monoclone with halogenase gene have been founded.We successfully construct a method to screen the monoclone with halogenase gene and aromatic polyketide synthases,heterologous expression of the environmental DNA gene in the monoclone,and analysis of the fermentation products through screening metagenomic libraries of soil by sequence-driven screening.The study lays a solid foundation for further study of the halogenase genes and its possibility of new type of natural halometabolites,and also provids a new way for the study of the drug discovery and the functional genes in microbial resources.