美达霉素产生菌中立体专一性酮基还原酶基因med-ORF12的表达  

Expression detection of med-ORF12 encoding a stereochemical ketoreductase possibly involved in medermycin biosynthesis

作　　者：孙锐[1,2] 刘明[1,2] 宫彩霞[1,2] 王蔚[1,2] 曾爱兵[3] 李爱英[1,2] 

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]华中师范大学农药与化学生物学教育部重点实验室,武汉430079 [3]温州医学院生命科学学院,温州325035

出　　处：《微生物学报》2012年第1期60-68,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30770036;31170050);浙江省自然科学基金(Y3110050);中央高校基本科研业务费(CCNU10A02011)~~

摘　　要：美达霉素是链霉菌产生的具有强抗肿瘤活性的芳香聚酮类抗生素,其砒喃环并内酯结构对于其抗癌活性非常重要。位于美达霉素生物合成基因簇中的基因med-ORF12编码立体专一性酮基还原酶,可能参与美达霉素的砒喃环并内酯结构中手性中心(C3S)的形成,但在美达霉素产生菌中的功能和表达还未曾研究。【目的和方法】为了研究med-ORF12在野生菌中的表达情况以及与美达霉素生物合成的关系,本文采用了原核表达、抗体制备、免疫杂交等技术方法对这个基因展开了体内表达研究。【结果】首先利用pET载体建立了med-ORF12的原核表达系统,在优化诱导表达条件的基础上获得了可溶性目的蛋白,制备了相应的多抗血清;然后利用多抗血清对美达霉素产生菌中的基因med-ORF12的表达情况进行了检测,表明在美达霉素产生菌中参与次生代谢的med-ORF12在稳定期大量表达,同时伴随美达霉素的大量积累。【结论】这些结果表明在美达霉素产生菌中,基因med-ORF12参与次生代谢,其表达与美达霉素生物合成有一定相关性。Medermycin, an aromatic polyketide antibiotic produced by streptomyces, possesses a stereochemical-pyran-ring lactone critical for its strong anticancer activity. The med-ORF12 located in the biosynthetic gene cluster of medermycin encodes a stereochemical ketoreductase. Based on many indirect data, we proposed it to be involved in the enantioselective reduction at C-3 in the formation of the pyran ring of medermycin. The direct genetic evidence about the function of med- ORF12 in the medermycin-producing strain has yet to be obtained. Enzymatic features, expression and regulation pattern of Med-ORF12 in the medermycin-producer still remain obscure. [ Objective and Methods] The present study aimed to investigate the expression profiles of med-ORF12 and relationship between Med-ORF12 and medermycin accumulation in medermycin-producers using prokaryotic expression, protein purification, polyclonal antiserum preparation, western blot. [ Results] First, we established a prokaryotic expression system of med-ORFt2 using a pET vector and optimized the induction conditions to accumulate the soluble Med-ORF12. Subsequently, we acquired the polyclonal antiserum against Med-ORF12 by immunizing the New Zealand rabbit with the purified protein. Finally, we detected the expression pattern of med-ORF12 in the medermycin producers with the obtained polyclonal antiserum, and found that med-ORF12 could express with a fairly high amount during the late stationary phase of the medermycin-producers, consistent with the accumulation of medermycin as a secondary metabolite. [ Conclusion] These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.

关 键 词：链霉菌 次生代谢 美达霉素 立体专一性酮基还原酶 

分 类 号：Q78[生物学—分子生物学]