在分枝杆菌中从简单碳源自给自足合成雄甾烯酮的研究

To self-sufficiently synthesize steroids from a simple carbon source in microorganisms is the direction to reform the processing system of steroidal drugs in pharmaceutical industry, which is now in the period of concept construction and model verification. On the basis of one of the existing industrial processing system of steroidal drugs, that is the microbial transformation of sterols to androstenones, we intend to achieve the self-sufficient biosynthesis of androstenone from a simple carbon source in a mycobacterial chassis by the philosophy and techniques of metabolic engineering and synthetic biology. Mycobacteria is a kind of classical bacteria used in the transformation of sterols to androstenones in industry, which is selected as the chassis because of its powerful capacity of transformation of sterols to androstenones and its existing DOXP/MEP metabolic pathway for diterpene biosynthesis. In a mycobacterial chassis, two metabolic modules for the biosynthesis of squalene and sterols will be constructed by the introduction of exogenous genes to dock with the metabolic pathways of transformation of sterols to androstenones and DOXP/MEP. In this project, the construction of metabolic module for the biosynthesis of sterols is the main work. The target sterol molecular to be synthesized has been determined as cholesterol because its biosynthesis pathway is shorter and easier to be constructed in contrast with it of the other alternative sterols. It has been clear that the biosynthesis of cholesterol from squalene involves 11 enzymes that catalyze 12 reactions. The main challenges in this project are the compatibility of foreign genes with the chassis, the balance of genes expression with the constructed metabolic flux, the coordination among different modules, and the possible interference of origin metabolism in the chassis with the introduced metabolic systems. Therefore, to screen and evolve the target genes, to optimize the regulatory pathways, and to remove the interference reactions in chassis will be the main methods to achieve the working objective.

在微生物中从简单碳源出发自给自足合成甾体药物，是甾体药物生产技术体系革新的大方向，目前正处于概念创立和模型验证时期。申请者基于"微生物转化甾醇为雄甾烯酮"这一条现有甾体药物生产技术路线，结合代谢工程/合成生物学的理念和方法，拟以该路线常用菌"分枝杆菌"为底盘，借用并修改该菌原有DOXP/MEP二萜合成途径，引入外源基因，在底盘中构建"三萜鲨烯合成模块"和"甾醇合成模块"，实现与该菌自身"甾醇转化为雄甾烯酮代谢模块"的对接，从而实现在该菌中从简单碳源自给自足合成雄甾烯酮。本项目的主要工作在于构建甾醇合成模块，经可行性分析，确定以胆固醇为目标甾醇，该模块从鲨烯到胆固醇的完整代谢，共涉及11个酶12步反应。该项目的难点在于外源基因与宿主的适配性、基因表达与代谢流的平衡性、模块运作的协调性及底盘的干扰代谢，因此基因/调控等元件的筛选进化、调节通路的优化和底盘干扰代谢的净化将是完成本项目的主要手段。

“甾醇微生物转化”制备甾体药物中间体已成为甾体制药工业的主流技术。本项目基于此技术体系，探索了在微生物中从简单碳源出发直接合成甾体药物的可行性，具体措施是在具有甾醇转化能力的微生物中创建甾醇合成途径，对接原有的甾醇转化途径，来实现甾体药物在微生物底盘中的全合成。主要研究内容可分为三个部分：一、为了在微生物中实现甾醇的全合成，组建了“鲨烯合成代谢模块”和“甾醇合成代谢模块”，涉及24个基因的筛选和优化，构建成功可生产角鲨烯和羊毛甾醇的工程微生物。二、为了创建高品质的雄甾烯酮工程底盘，针对常规雄甾烯酮生产菌副产物多和降解率高等共性难题，开展了甾醇降解关键机制的研究，揭示了侧链不完全降解副产物C22羟基孕甾的生成机理以及母核降解的相关机制，提出了“甾醇代谢生产雄甾烯酮模块”的构建策略，实现了生产高品质雄甾烯酮底盘的创建，结果表明这些工程微生物可以在水相体系中转化50g/l甾醇，转化率>95%，收率（w/w）>45%，技术指标国际领先。三、通过底盘净化和启动子工程等优化手段，完成了外源“鲨烯合成代谢模块”和“甾醇合成代谢模块”在分枝杆菌底盘中的组装，检测到了雄甾烯酮的生成，达到了预期目标。本项目已发表6篇SCI论文，申请了2项国际专利，其中一项已在国内、日本、欧洲和美国进入国家审核阶段。本项目所创建的高品质雄甾烯酮工程菌作为主要内容之一，获得了2017年度中国石油和化学工业联合会技术发明二等奖。本项目的科学意义与应用价值在于：一、证实可以在微生物中实现从基础碳源到甾醇再到雄甾烯酮的合成，为开发具有实用价值的甾体药物全合成微生物细胞工厂提供了参考；二、率先揭示了分枝杆菌代谢甾醇的一些关键机制，论证并命名了雄甾烯酮代谢途径之外的一条新的C22-羟基孕甾代谢途径；三、开发了系列具有自主知识产权的工程微生物，其中C22-羟基孕甾和雄甾烯酮9-OHAD的工程菌已在企业得到了应用。