Production, Separation And Structural Identification Of Secondary Metabolites Produced By Streptomyces

Secondary metabolites producing by Streptomyces play important role in natural product drugs. Secondary metabolism is the process that microorganisms in certain period of growth (usually in stationary phase) synthesize a number of products with some uncertain functions from primary metabolic precursor. These process are carried out by a serial of enzymatic reactions in microorganisms using primary metabolites. Based on the genome sequencing of microbe and bioinformatics analysis, some gene clusters of other small molecules were elucidated in the microbial chromosomes. The production of these small molecules is usually very low or even can't be detected during the fermentation. However, some structurally novel compounds were generated or the production was improved by heterologous expression of the biosynthetic gene clusters. It benefits us to discover-more novel compounds.Three parts are discussed in this dissertation, including 1) Fermentation of Streptomycete rochei ATCC10739, separation and isolation of secondary metabolite Borrelidin,2) Heterogenous expression of some small secondary metabolites' gene clusters from Streptomyces TP-A2060, and 3) Heterogenous expression of some small secondary metabolites'gene clusters from Streptomyces 371.Borrelidin is a structurally distinct 18-membered macrolide antibiotic isolated from Streptomycete rochei ATCC10739, which was found to inhibit cyclindependent kinase (CDK) and show antiangiogenesis activity (IC50=0.4 ngmL-1). Our work is to optimize fermentation condition, separate and isolate this compound, and confirm its structure. We optimize the fermentation conditions in order to stabilize and increase borrelidin's production. The optimal fermentation conditions were determined as follow:the ISP-2 medium with 1% glycerol; initial pH 6.0; culture temperature,30℃; relative humidity,50% and fermentation time,7 days. The structure of Borrelidin was confirmed by ESI-MS, HR-ESI-MS and 1HNMR in comparison with published rusults.Streptomyces TP-A2060 is the producing strain of antitumor antibiotic yatakemycin. In this part, senven gene clusters involved in biosynthesis of five terpenoids, a polyketide compound and an unknown compound from this strain were heterogenous expressioned. We cloned these gene clusters, inserted the DNA fragments into expressing vectors, and tried to transfer the expression plasmid into heterogenous host Streptomyces coelicolor CH999, now we obtained all expression plasmids and two mutant strains of Tep-1 and Tep-4. Two mutant strains of Tep-1 and Tep-4 were fermented. We hope we could get the rest of mutant strains through protoplast transformation soon. The expectant secondary metabolites are isolating through fermentation of mutant strains. The works need further exploration.Streptomyces 371 is the producing strain of biopesticide aureonucleomycin. We try to get three terpenoids and a polyketide compound by heterogenous expression strategy. Now, we had obtained all expression plasmids and mutant strains of Tep-7 and Pkz, and two secondary metabolites of an expectant terpenoid and a polyketide compound were detected by HPLC-MS from the fermentation broth. The isolation and structure eludication of these compounds are on the way.