吡咯烷抗生素茴香霉素的生物合成机制研究

The protein synthesis inhibitor anisomycin features a unique pyrrolidine system and exhibits diverse biological and pharmacological activities. Anisomycin is isolated from Streptomyces hygrospinosus var. beijingensis. Owing to its reversible 60S ribosomal subunit binding, anisomycin can block peptide bond formation and shows potent selective activity against pathogenic protozoa and fungi. It is one of the important effective components in Agricultural Antibiotic 120, which has been widely used to treat the crops decay in China. However, its biosynthetic origin has remained obscure for over 60 years. In this project, the anisomycin biosynthetic gene cluster was cloned, several anisomycin biosynthetic gene deletion mutants were constructed, six accumulated intermediates were characterized, and enzymatic reaction system of dehydrogenase AniN was established. Based on these results, we will perform a series of combination experiments including in vivo genetic manipulation, isotopic labelling and in vitro biochemical assays to elucidate the mechanism of the pyrrolidine formation in the anisomycin biosynthetic pathway. We will reveal a novel pathway for biosynthesis of pyrrolidine that is strikingly different from the previously characterized nonribosomal peptide synthetase pathways, in which a cryptic glycosylation reaction is involved in the biosynthesis of anisomycin. The research results will provide insight into the mechanism of a new dehydrogenase, and will obtain an effective genetic element used in the structural innovation of drug molecules through synthetic biology strategy.

蛋白质合成抑制剂茴香霉素是一个具有独特的吡咯烷结构和良好的生物活性的微生物来源的天然产物。茴香霉素由刺孢吸水链霉菌北京变种产生，是农抗120的活性成分之一，但发现了60多年的茴香霉素，其生物合成途径至今仍然不清楚。本项目在鉴定茴香霉素生物合成基因簇，构建茴香霉素生物合成基因突变株，分离鉴定多个中间体基础上，通过基因回补、中间物喂养、同位素标记以及体外酶学分析，研究确定脱氢酶AniN在催化吡咯烷环形成中的作用，丰富对自然界脱氢酶的理解。通过解析茴香霉素生物合成途径，为农用抗生素-农抗120的品种和品质改造建立基础。系统地研究葡萄糖基与吡咯烷形成的关系，以及脱氢酶AniN的底物专一性，预期对一类新的吡咯烷形成机制的系统阐述，将获得一个有效生物合成元件用于合成生物学进行相关药物分子的结构创新。

茴香霉素是一种具有抑制蛋白质合成活性的吡咯烷生物碱，因其具有多种生物和药理作用而得到广泛的应用，但茴香霉素发现至今60余年，它的生物合成机理一直是个未解之谜。本研究采用生物活性为导向的高通量筛选技术，克隆到了茴香霉素生物合成基因。组合运用生物信息、体内遗传和体外生化分析，鉴定表征了一个新颖的吡咯烷生物合成途径负责茴香霉素的生物合成。我们鉴定了4个核心基因负责茴香霉素吡咯烷的生物合成。包括氨基转移酶AniQ催化两步转氨反应，转酮酶AniP催化4-羟基苯丙酮酸和3-磷酸甘油醛的缩合，糖基转移酶AniO催化隐藏的糖基化反应，兼具氧化和还原双功能的脱氢酶AniN介导吡咯烷的形成。研究发现一个隐蔽但至关重要的糖基化反应，意想不到的转胺反应，以及由单一酶催化的多步反应负责完成吡咯烷骨架的组装。通过体内遗传和体外生化实验，也阐明了AniF作为正调控基因在转录水平调控茴香霉素生物合成。本研究也发现一个乙酰转移酶AniI能催化吡咯烷环上羟基的酰化反应，并能耐受多种酰基供体，可以催化乙酰、丁酰和异戊酰基基团的加载。通过强启动子过表达aniI，也促进了茴香霉素及其类似物的高产。这些研究为新型吡咯烷类天然产物的基因组挖掘提供了机会，也为通过工程化茴香霉素的生物合成进行吡咯烷类抗生素的药物开发奠定了基础。