刺糖多孢菌中多杀菌素生物合成关键代谢节点的解析及工程改造

Spinosad, produced by Saccharopolyspora spinosa, is an important macrolide antibiotics with potent insecticidal properties. As a high efficiency, safe and environment-friendly pesticide, spinosad has been widely used in agriculture. However, due to the low fermentation yield, the industrial production of spinosad is quite limited in China. In addition, although many previous works have been done for spinosad overproduction, the problem is still challenging. Therefore, this project aims to reveal rate-limiting steps in spinosad biosynthesis and to improve the production of spinosad. Firstly, based on our previous work, the Saccharopolyspora spinosa-derived cell-free system will be set up. Then, the biosynthesis of spinosad in S. spinosa can be quantitatively analyzed by using the cell-free system. Briefly, the biosynthetic process of spinosad is divided into two stages: the biosynthesis of polyketide and post-PKS modification. Each component involved in polyketide biosynthesis, such as precursors, NADPH and acetyl-CoA carboxylase etc., can be monitored and quantitative analyzed by radioactive assay. On the other hand, the kinetic parameters of substrates and enzymes, which involved in post-PKS modification (such as glycosylation and methylation etc.) also can be investigated by using LC-MS methods. Lastly, based on the above information, the metabolic nodes will be revealed and engineered to enhance the fermentation production of spinosad. The directly quantitative investigation of spinosad biosynthesis will help to develop a more reliable engineering strategy for spinosad overproduction. The strategy developed by our research may also be useful to improve other antibiotics production.

多杀菌素是由刺糖多孢菌产生的具有广谱杀虫活性的大环内酯类抗生素。作为一种高效、低毒、环境友好的“绿色”抗生素，多杀菌素被广泛使用。由于目前国内菌株发酵单位低，而前期进行的诱变以及基因改造尝试，仍然无法与陶氏公司的高产菌株竞争，整个市场被国外产品垄断。为此，基于本研究室前期的工作基础，本项目拟首先建立刺糖多孢菌的Cell-free系统；其次基于该系统，一方面利用14C标记的同位素实验考察PKS合成过程中的制约因素（如底物、氧化还原力及关键酶等）；另一方面通过制备关键中间体和合成途径中的蛋白，利用LC-MS监测PKS后修饰过程（如糖基化、甲基化等）并分析其中的限速步骤；最后通过对限速步骤进行改造实现多杀菌素的高效合成。预期通过该研究能找到多杀菌素合成途径中的代谢瓶颈并指导随后的代谢工程改造，有望大幅提高多杀菌素的发酵水平，而且本研究提出的研究思路和方法对其他抗生素代谢工程改造有良好的借鉴作用。

多杀菌素(Spinosyn)是一种由刺糖多孢菌(Saccharopolyspora spinosa)产生的大环内酯类抗生素。作为一种高效、低毒、环境友好的“绿色”杀虫剂，多杀菌素具有极大的开发价值。由于发酵周期长、产量低，前期相关的工程改造虽提高了多杀菌素的发酵水平但仍无法满足需要，极大限制了我国多杀菌素的生产，导致其国内市场长期为国外跨国公司所垄断。本研究通过建立刺糖多孢菌的细菌人工染色体文库，即BAC(Bacterial Artifical Choromosome)文库，筛选并获得包含完整多杀菌素全基因簇的BAC质粒，命名为C4I6。随后利用Red/ET技术将链霉菌接合转移、遗传筛选标记、整合位点等相关元件插入C4I6质粒中，获得质粒C4I6-M。然后利用E.coli ET12567(pUB307)，通过三亲本接合转移将C4I6-M质粒分别整合到白色链霉菌J1074(Streptomyces albus J1074)和变铅青链霉菌TK24(S. lividans TK24)。本研究随后利用高分辨质谱(High resolustion Mass spectrum)在两株异源链霉菌宿主中均检测到了微量的多杀菌素。为进一步提高多杀菌素的异源合成效率，本研究建立了基于高分辨质谱的链霉菌蛋白质组和代谢组学分析方法。通过组学分析发现了多个可能制约多杀菌素异源合成的限速步骤，包括糖基供体的合成、后修饰、PKS的合成等。随后通过酵母组装方法，利用链霉菌强启动子逐个对各个限速模块进行优化。目前通过对糖基合成等催化模块的优化使得多杀菌素在链霉菌中的发酵产量提升了近1000倍达到1640μg/L，后续通过其他代谢节点的的优化理应可大幅提高多杀菌素的异源合成效率。本研究首次实现了大型聚酮类化合物多杀菌素在链霉菌中的异源合成，同时利用组学分析方法系统而高效的指导了多杀菌素的代谢工程改造，该研究一方面为后续进一步提高多杀菌素发酵水平直至实现其国产化奠定了基础，同时本研究开发的相关的工具和研究策略可应用于其他新药研发。