Studies On The Secondary Metabolites Of Deep Sea Metagenomic Clones

In the study of microbial metabolism and biosynthesis pathway, the vast majority of microorganisms are difficult or cannot be obtained in pure culture by cultivation methods. As a result, we cannot recognize the diversity of microbial genes which encode the biosynthesis of metabolites comprehensively as well as the species in nature. Therefore, culture-independent methods are the key way to study the genetic and metabolite diversity of microorganisms. By extracting the total DNA of microorganisms directly from environmental samples and cloning into the appropriate educable host cells, utilizing metagenomics strategies to study the genetic constitution of microorganism contained in environmental samples have become an important analytical method to analyze aggressive environmental samples such as marine samples.In this study, chemical screening of17clones, derived from deep-sea sediments metagenomic libraries and possessed cytotoxic activity, was carried out. After comparing with the host E. coli, clones10-1and25D7could produce special metabolites. Large-scale fermentation of both E. coli host and10-1were carried in-depth study, including metabolite extraction, isolation and structure identification.18compounds were identified as6indole alkaloids,5chloramphenicol derivatives and7cyclic peptides.3-hydroxy-2-indolone compounds and indirubin produced by10-1were special metabolites and the biosynthetic pathway may be the substrate indole within the function of dioxygenase gene oxidized to isatin and then condensed to indirubin. Cultivation with the substrate5-bromoindole confirmed the biosynthetic pathway. Study of clone25D7metabolites conducted through addition of substrate5-bromoindole into the culture directly. With the guidance of LC-MS,4brominated products were isolated from the fermentation. The biosynthetic pathway of the new brominated bis-indole derivatives confirmed to be the expression of monooxygenase and dioxygenase functional genens in the clones to produce isatin and7-hydroxy indole, further aggregated to the new compounds. According to the study,23compounds were obtained from two strains of deep-sea metagenomic clones and host E. coli fermentation, including two new compounds; verified the biosynthesis pathway of four special metabolites, laid a foundation for further development and utilization of deep-sea functional genes.