西索米星生物合成基因sis I功能的研究

The functional exploration of sisIin sisomicin biosynthesis gene cluster

通过生物学试验,构建依纽小单孢菌TS388(Micromonospora inyoensis TS388)中sisI基因缺失工程菌,再通过分析其次级代谢产物的变化,揭示西索米星生物合成基因簇上sisI基因的功能.首先构建用于sisI基因框内敲除的穿梭载体pKTI3,经接合转移导入依纽小单孢菌TS388中,通过影印筛选及PCR鉴定获得sisI功能缺失工程菌TI1102(△sisI).工程菌经发酵,提取代谢产物,再经TLC及MS分析,用以比较亲株与工程菌代谢产物结构的差异.结果显示:工程菌TI1102代谢产物结构发生变化,不再合成西索米星,主要积累中间代谢产物JI-20A,由此证明了sisI基因参与了JI-20A到西索米星的3′,4′-双脱羟基作用.获得主产JI-20A的工程菌为该类半合成药物提供了重要前导化合物.

The sisI deletion mutant was constructed in M.inyoensis TS388 by the biological experiments and the function of sisI in sisomicin biosynthetic gene cluster was revealed by analyzing the changes of its secondary metabolites. First, the recombinant plasmid pKTI3 used for the sisI in-frame delection was constructed and then was introduced into M. inyoensis TS388 by conjugation. The sisI-disruption mutant M. inyoensis TI1102 （AsisI） was screened out by replica plating and PCR validation. Metabolites which was isolated from the fermentation broth of TI1102 was analyzed by TLC and MS to compare the differences of metabolite structure between the parent strain and engineering strain. The result indicated that the structure of metabolites of the mutant strain TI1102 is changes, and TI1102 mainly accumulates intermediate metabolite JI-20A instead of sisomicin. It was demonstrated that sisI might be responsible for double dehydroxylation at C-3＇ and C-4＇ of JI-20A to sisomicin. An engineered bacteria that mainly produces monocomponent JI-20A was obtained, which provides the important lead compounds for semi-synthetic drugs.