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

摘要:

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。

Abstract:

Abstract: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-ORF12 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．