Preliminary Research Of Six Potential Gene Clusters Of Amycolatopsis YIM 96748 By Heterologous Expression

The primary sources of pharmaceutical research and development are from natural products which mainly provided by secondary metabolite by actinomycetes.With the change of natural environment and overuse of antibiotics,new epidemics continue to break out,the pathogenic bacteria became superbugs and drug-resistant bacteria by evolution,it is no time to delay the development of new medicines.The genome of extremophilic microbes become fancy in the evolutionary process because of special environment,thus they also own novel natural products biosynthetic gene clusters.However it is difficult to cultivate them and performing genetic manipulation,and it is hard to utilize their genes directly.In this study,Amycolatopsis YIM 96748 derived from saline environment was used as the research material.Identifying total gene clusters by bioinformatics analysis.Six high-potential biosynthetic gene clusters were selected for heterologous expression by comparing with the existing genomes and known gene clusters in the database,named clusters 2,9,11,19,22,30,respectively.A cosmid genomic library belonging to Amycolatopsis YIM 96748 was successfully constructed,containing 1800 clones covering the entire genome.Designing and making specific screening probes,using Colony hybridization and immunochromogenic technology to screen the target gene clusters from the library.The six complete gene clusters were transferred to the four heterologous expression host bacteria Streptomyces.albus J1074,Streptomyces.coelicolor M145,Streptomyces.lividans TK24 and Streptomyces.lividans SBT18 through conjugation,respectively.Successfully constructed 25 heterologous expression mutants,thirteen strains of which were fermented,and fermented extracts were tested and analyzed.This work promotes the utilization of genetic resources of halophilic bacteria.The system of heterologous expression in vitro of halophilic Amycolatopsis YIM 96748 has been preliminarily established,which set the tone and provide a platform for later in-depth research.