High Throughput Screening Of Halophilic Actinomycetes Based On Functional Genes And Heterologous Expression Study Of Potential Strains

Novel drug discovery need to be accelerated as drug-resistant bacteria increase and new diseases emerge gradually. Drugs from microbial were the major source of new drug discovery. Microbial from extreme environment have became the hot spot for drug discovery as microbial from normal environment have been well studied. Some active secondary metabolites have been found from halophilic actinomycetes. Plenty of halophilic actinomycetes strains have been accumulated, but the research on its natural products was slow because of some shortcomings such as fermentation difficulties.A strain library containing500halophilic actinomycetes strains was constructed and genomic DNA of each strain was isolated respectively. Four types of natural products key genes were selected and digoxin labeled probe of each gene was prepared by nest PCR using degenerate primers. These primers were used for high throughput screening against the genomic DNA library. As a result, plenty of strains containing different key genes were obtained and31of them were selected for further study as they contain different type of key genes simultaneously. These strains could produce various types of natural products and some of them might be new structure compounds.Two strains, YIM96935and YIM96633, which contain many types of key genes, were chosen for genomic sequencing. After bioinformatics analysis,5and16possible biosynthetic gene clusters were found from each strain. Some possible clusters showed certain similarities with known gene clusters which have proven to code different types of natural products, these compounds have good anti-tumor, antioxidant, antibacterial, anti-inflammatory and immune inhibitory activities. Most possible clusters showed low similarities with known gene clusters, which could code new structure natural products. Pilot study of heterologous expression was carried out for the two sequenced strains.In this study, high throughput screening method fit for halophilic actinomycetes strains was constructed and high potential strains was obtained. Bioinformatics analysis results proved the efficiency of this method. This method could partially solve the dilemma of discovery novel compounds from halophilic actinomycetes.