Functional Screening And Activity Identification Of Bioactive Substances In Soil Metagenomic Library

Soil was such a diverse ecosystem,that millions of microbes were contained in just one gram of soil.Microbe was one of the main sources of bioactive compounds,including antibiotics,anticancer drugs and biological enzymes.However,only<1%of microbes could be cultured under standard laboratory conditions.This greatly limited the discovery of microbial natural products.So it is urgent to develop a new method and train of thought for the discovery of microbial natural products.Metagenomics technology,did not dependent on the microbial culture technology.By metagenomic technology,the microbial DNA was extracted directly from environmental samples,and the total DNA was purified and concentrated to obtain high quality DNA.Then the repaired DNA was connected to suitable vectors(plasmid,cosmid,fosmid and BAC),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 to discover new products.In this study,we constructed a soil metagenomic library by using the method of direct lysis.The DNA from the soil environment in the library was transferred to the host of Streptomyces by means of the high efficient expression system of Streptomyces.We selected positive clones which have antibacterial activity based on their phenotype and fermented them.The fermentation product was collected and the chemical structures of the compounds were expected to be studied.In addition,the effect of crude extract on the inhibition and clearance effect of Staphylococcus aureus biofilm was studied.Specific results are as follow:1?The metagenomic library of Emei Mount,Sichuan was constructed successfully,which contained 200,000 clones with an average insert of 33.7 kb.After testing,the library diversity is good.2?The strains were transferred to Streptomyces albus,Streptomyces coelicolor,and Streptomyces lividans,by observing the morphology and pigment production after the successful conjugation,not positive clones were obtained.By the detection of antibacterial activity,the positive clones No.110 and No.123 were obtained with the successful transfer of Streptomyces albus.3?Two active clones were obtained,whose fermentation extracts was detected by the high performance liquid chromatography,and showed obvious different peaks compared to the control groups.Furthermore,their fermentation extracts were studied for the inhibitory effect on Staphylococcus aureus biofilm.Their fermentation products have a good inhibitory effect on the formation of S.aureus biofilm,and the inhibitory effect could exceed 90%when the concentration of sample is 2 MIC(minimmum inhibitory concentration,MIC).In addition,two samples have significantly clearance effect on S.aureus biofilm,and the clearance rate of EM110 was higher than EM123.