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Production, Separation and Structural Identification of Secondary Metabolites Produced by Streptomyces

Author: LiZhou
Tutor: ZhaoShengYin；TangGongLi
School: Donghua University
Course: Organic Chemistry
Keywords: Natural Products Whole genome sequencing Bioinformatic analysis Borrelidin Terpenoids
CLC: TQ465
Type: Master's thesis
Year: 2011
Downloads: 103
Quote: 0
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Abstract


Source of secondary metabolites of Streptomyces is an important part of the natural product drugs, the so-called secondary metabolites of microorganisms in certain growth period (generally stable anagen), to the primary metabolite of the precursor synthesis of some of the microbial life activities do not have a clear function of the substance of the process. Primary metabolic product of small molecules to the reaction substrate, a specific series of enzyme protein catalytic reaction, i.e., a plurality of different functions of the enzyme in turn effect, finally obtained microbial secondary metabolites. By whole microbial genome sequencing and bioinformatics analysis found that the microbial chromosome biosynthetic gene cluster of many small molecules, these small molecule compounds in the fermentation process yields are low, or not, by the heterologous expression The fermented get some structurally novel small molecule compounds or compound yield improved, and this enables us to find more new compounds. This topic is divided into three parts: 1) Streptomyces Streptomyces rochei ATCC10739, fermentation, the secondary metabolites Borrelidin Purification and structure confirmation; 2) Streptomyces Streptomyces TP-A2060 strains of several small molecule secondary metabolite gene clusters heterologous expression; 3) Streptomyces strains of Streptomyces 371, several small molecules from the secondary metabolites of the heterologous expression of the gene cluster. Borrelidin from Streptomyces (Streptomyces rochei ATCC 10739) isolated from the seed 18 membered macrolide antibiotics, having the CDK (on the cell cycle-dependent protein kinase) inhibitory activity and activity of inhibiting angiogenesis. Our main work is to optimize the fermentation conditions and the compounds were isolated and purified, conclusive evidence of its structure. Optimization of the fermentation conditions in the fermentation process, in order to stabilize and improve the yield of compound. Ultimately determine the optimum fermentation conditions: ISP-2 medium supplemented with 1% glycerol, initial pH 6.0, culture temperature 30 ° C, 50% relative humidity, fermentation time for 7d. The compounds were characterized by ESI-MS, HR-ESI-MS and 1HNMR confirm their structure and literatures. Streptomyces TP-A2060 is antitumor antibiotics Yatakemycin, produce bacteria, several small molecule compounds genes for heterologous expression. By whole genome sequencing and bioinformatics analysis found that the compound including five terpene compounds, an aromatic polyketides, one can not predict the structure type of compound. By cloning plasmid, the heterologous expression Construction of plasmid and the mutant strain, we successfully obtained the entire gene cluster heterologous expression plasmid, Tep-1 and Tep-4, a mutant strain, a small amount of the fermentation and the two mutants . I hope the rest of the mutant obtained by protoplast transformation, the final fermentation to give the desired compound. Further Construction of mutants and fermentation is still a work in progress. Streptomyces 371 is of biological pesticides gold generation of nuclear neomycin bacteria, the work we have done three terpene compounds possible and an aromatic polyketides through heterologous expression. The entire gene cluster has been successfully obtained heterologous expression plasmid, TEP-7 and Pkz mutant, mass spectrometry results for the two mutant strains of a small amount of the fermentation expected terpenes and polyketide. Further research work is still in progress.

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