多杀菌素及其衍生物的合成生物学研究

Spinosad and its analogs produced by Saccharopolyspora spinosa are a serial promising macrolide insect control agents produced by Saccharopolyspora spinosa. Spinosad was awarded with the ‘Presidential Green Chemistry Challenge Awards’ of US for its high efficacy and low toxicity properites. The most tough problem needs to be solved is the difficulty of genetic manipulation and the instability of the fermentation. This project is focusing on the study of manufacture of spinosad and its analogs based on synthetic biology approach. The biosynthetic gene clusters responsible for spinosad will be assembled in a shuttle BAC plasmid by Gibson Assembly, and introduced to several heterologous strains such as Streptomyces avermitilis, S. coelicolor and S. venezuela which have been optimized as appropriate chassises for spinosad expression. Multiple omics analysis, computational biology study will identify the strategy for module improvement. Compatibility of the synthetic microbial systems will be studied through codon optimization, promoter optimization, RBS engineering, construction of regulatory modules and improving the modularity of each parts. We aim to construct 1 or 2 new microbial systems for heterologous production of spinosad and obtain 3 to 5 new analogs. This project will not only fit the demand of agricultural production and fermentation industry in China, but also the theoretical issues of synthetic biology. In addition, it will enrich and improve the research framework of synthetic biology in China and realize technological innovation in related areas.

多杀菌素是由刺糖多孢菌产生的一类新型大环内酯类农用抗生素，因高效低毒而获得美国“总统绿色化学品挑战奖”。目前我国仍未突破其技术垄断实现国产化。刺糖多孢菌遗传操作困难、产素稳定性差是关键问题之一。本项目以多杀菌素及其衍生物的合成生物学研究为主题，拟应用Gibson assembly方法构建多杀菌素生物合成基因簇并导入到背景清晰的适合多杀菌素表达的底盘细胞（如阿维链霉菌、委内瑞拉链霉菌、天蓝色链霉菌）进行异源表达；经过组学分析、计算生物学研究确定模块的改造策略；通过密码子优化、启动子优化、RBS改造、调控模块构建、提高各元件模块化性能等进行合成微生物体系的适配性研究；最终集成产生多杀菌素的新微生物体系1-2个、结构得到修饰的新多杀菌素衍生物3-5个，为我国多杀菌素的产业化奠定基础，也为进一步丰富和完善合成生物学研究框架、实现相关领域的技术创新进行探索。

多杀菌素是由刺糖多孢菌产生的高效低毒杀虫药，本项目进行了多杀菌素的合成生物学研究，成功将多杀菌素生物合成基因簇克隆至 pCC1BAC 载体中，进行了多杀菌素鼠李糖合成相关基因的克隆，再利用red/ET重组技术改造BAC载体，得到了可用于大肠杆菌-链霉菌结合转移的穿梭BAC载体pSETBAC::SPINOSYN，用结合转移的方法，将上述载体转入阿维链霉菌、白色链霉菌、天蓝色链霉菌等宿主中，但是经过多次尝试，均未获得转化子。因此调整了研究方案，从微生物资源入手，通过单菌多产物、表观遗传调控、共培养等多种方法，有效提高微生物次级代谢产物的多样性，从中筛选具有杀虫等活性的微生物天然产物。我们从1株真菌中筛选获得了具有较好杀虫活性的天然产物C1、C2，LD50均为12.5µg/ml；C7、C8，LD50均为25µg/ml；有望成为杀虫新药的候选化合物。此外还筛选获得了具有抗真菌、抗病毒等活性的微生物天然产物，为微生物来源的新药发现奠定了基础。