动态代谢组指导的冰城链霉菌米尔贝霉素关键代谢途径改造

Milbemycins produced by Streptomyces species is a class of important agricultural antibiotics with high effectivity, low toxicity, and environmental protection. Hence, it is urgent to improve the production of milbemycins to meet the requirements of extensive application for large crops. Streptomyces bingchenggensis is a good producer of milbemycins screened by our team, and it has been completed the whole genome sequencing. In this project, we will carry out the basic researches of synthetic biology about the dynamic metabolome guided engineering of key metabolic pathways for milbemycins biosynthesis in Streptomyces bingchenggensis. First, dynamic metabolome analysis will be used for the systematic identification of the key targets (intracellular metabolites) influencing the productions of milbemycins from the global view of cell metabolism of Streptomyces bingchenggensis. Then, the critical biosynthetic pathways of these targets will be de novo designed and reconstructed with the genetic tools, such as promoters and insulators. Finally, an efficient experimental scheme to coordinate different reconstructed metabolic pathways will be designed by the central composite designs, and then a non-linear multiple regression model will be built by response surface methodology, which helps to determine the optimal transcription level of each crucial metabolic pathway, and further guide the promoter selection to enable the optimal coordination of key metabolic pathways. This project will not only deepen the recognition of important metabolic mechanisms of biosynthesis of milbemycins in Streptomyces bingchenggensis, but also provide a rational guidance for production improvement of milbemycins. This will further help us to obtain the high-yield Streptomyces bingchenggensis strains, which will lay an important foundation for the production improvement and further industrialization of milbemycins.

米尔贝霉素是由链霉菌产生的重要农用抗生素，因此，迫切需要进一步提高米尔贝霉素产量以推广其在大型作物中的应用。本项目拟以本课题组完成全基因组测序的冰城链霉菌为研究对象，开展动态代谢组指导的冰城链霉菌米尔贝霉素生物合成关键代谢途径改造的基础研究。首先，利用动态代谢组学在冰城链霉菌全局代谢水平上，鉴定影响米尔贝霉素产量的关键代谢靶点；然后，利用链霉菌启动子、绝缘子等表达控制元件，重构关键代谢靶点的生物合成途径；最后，针对适配多条关键代谢途径设计中心复合试验，通过响应面分析建立多元非线性回归模型，模拟菌株产量最高时各关键代谢途径的最佳转录水平，进而指导选择合适强度的启动子控制相应代谢途径的转录，实现对关键代谢途径的最优化适配。本项目能够促进对冰城链霉菌米尔贝霉素生物合成关键代谢机制的深入认识，并为提高米尔贝霉素产量提供理性指导，将为进一步获得高产工程菌株，实现米尔贝霉素产业化奠定重要的研究基础。

米尔贝霉素是由链霉菌产生的重要农用天然产物，迫切需要进一步提高工程菌株的米尔贝霉素产量以推广其在农业领域中的应用。冰城链霉菌是本实验室筛选得到的一株优良米尔贝霉素产生菌。为了高效构建米尔贝霉素高产工程菌，本项目拟利用比较代谢组学手段，鉴定影响冰城链霉菌米尔贝霉素生物合成的关键靶点；进一步，通过建立代谢途径的适配策略对关键靶点进行优化改造，实现米尔贝霉素生物合成途径的优化与菌株产量的提高。.聚焦该任务，本项目建立了冰城链霉菌动态代谢组学分析方法，基于比较代谢组学数据，鉴定了多个影响米尔贝霉素生物合成的关键代谢靶点；建立了链霉菌工程改造自调控策略，实现了米尔贝霉素高效生物合成途径的构建与菌株产量的显著提高。.本研究不仅获得了米尔贝霉素高产工程菌，也获得了放线菌天然产物农药高产工程菌构建所必需的合成生物学元件、方法与策略，形成了具有自主知识产权的放线菌天然产物农药高产菌研发平台，这将为其他天然产物药物高产工程菌开发提供重要支撑。