创新霉素生物合成途径的阐释和异源表达

创新霉素是中国发现的第一个全新结构的抗生素，对于大肠杆菌引起的败血症、胆囊炎和尿路感染等疾病有显著的疗效。创新霉素识别的靶标独特(细菌色氨酰tRNA合成酶)，且结构新颖，但生物合成机制还未阐明。前期工作对创新霉素产生菌-济南游动放线菌-进行了全基因组测序(预计2015年5月完成)，本项目拟对其基因组序列进行生物信息学分析，对可能的创新霉素生物合成基因簇进行大片段直接克隆，导入合适异源宿主细胞进行表达，鉴定基因簇的完整性；进而通过对关键基因进行失活、基因回补、体外酶反应以阐明创新霉素分子中硫的生源途径和吲哚并噻喃三环的合成机制；最后通过组合生物合成等手段产生新的衍生物用于进一步的药效评价。本项目有助于阐释创新霉素中稀有的含硫杂环体系的生物合成机制，也为将来的药物开发提供重要的基因资源。

Chuangxinmycin, as a novel indole alkaloids antibiotic, was firstly discovered in China and effective for treatment of septicaemia, urinary and biliary infections caused by Escherichia coli in preliminary clinical trial. It is featured with a rare structure (thiopyrano[4,3,2-cd]indole) and its ability to inhibit a special target (bacterial tryptophanyl tRNA synthetase). Up to now, the biosynthetic pathway of this antibiotic remains still obscure. The present project intends to clarify the biosynthetic pathway of chuangxinmycin : Firstly, we will screen the proposed chuangxinmycin biosynthetic gene cluster based on bioinformatic analysis of the genome of chuangxinmycin-producing Actinoplanes tsinanensis. Subsequently, using Red/ET recombineering, we intend to clone directly the complete gene cluster and transfer it into a heterologous host for efficient expression and identify the completeness of chuangxinmycin biosynthetic gene cluster. Then, we will clarify the biogenesis of sulfur in chuangxinmycin and the mechanism of thiopyrano[4,3,2-cd]indole formation by gene inactivation and in vitro enzymatic reactions. Finally, we plan to generate novel derivatives by combinatorial biosynthesis for further efficacy evaluation. Our investigations here will help elucidate the biosynthetic pathway of chuanxinmycin, representing a novel family of antibiotics, as well as enrich the gene resources for the novel thiopyrano[4,3,2-cd]indole formation and open a possibility to produce more engineered natural products used for drug development.