基于定量代谢组学分析指导的多杀菌素高效异源合成

Spinosad, produced by Saccharopolyspora spinosa, is an important type-I PKS-derived macrolide antibiotic with potent insecticidal properties. As a high efficiency, safety and environment-friendly pesticide, spinosad has been widely used in agriculture. However, due to the low productivity, 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. In this project, we firstly plan to construct a bacterial artificial chromosome (BAC) library of Saccharopolyspora spinosa. And the spinosad will be attempted to biosynthesize in Streptomyces host by using BAC plasmid that contains the whole spinosad biosynthetic gene cluster. Secondly, the metabolomics approach will be developed in Streptomyces spp. To reveal the rate-limiting steps involved in spinosad heterologous biosynthesis, the metabolomics approach (accompany with proteomics analysis) will be applied. And the metabolic engineering work can be carried out to promote the heterologous production of spinosad in Streptomyces. Lastly, the biosynthetic modules of spinosad will be optimized by in vitro reconstitution, and subsequently assembled by using a Streptomyces promoter library. These optimized biosynthetic modules will be introduced into the chromosome of Streptomyces host to further enhance the production of spinosad. Followed by several rounds of metabolomics-guided metabolic engineering, the production of spinosad will be gradually increased in Streptomyces. The successful of this project will lay the foundation for high-efficient biosynthesis of spinosad in Streptomyces, and the strategy and tools developed in this project may also be applied to dramatically improve the production of other antibiotics.

多杀菌素(I型聚酮)是由刺糖多孢菌产生的一种高效、低毒的“绿色”杀虫剂，具有极大的开发价值。经过多年对原始产生菌的诱变和遗传改造，虽然有很大幅度的提升但仍无法接近国外的水平，至今没有实现国产化。本项目拟首先建立刺糖多孢菌的BAC文库，利用BAC质粒初步实现多杀菌素在链霉菌中的异源合成；然后建立链霉菌代谢组学分析方法，通过定量代谢组学分析，辅之于蛋白质组分析，在代谢物和蛋白水平系统考察并确认制约多杀菌素合成的节点，通过相应的代谢工程改造提高多杀菌素的异源表达水平。而后针对多杀菌素各合成模块的特征，利用链霉菌启动子库实现各合成模块的优化和组装，促进多杀菌素的高产；这样的循环可以往复进行，从而循序渐进提高多杀菌素的产量。在此过程中运用发酵工程加以配合，充分对突变株的结果进行放大。本研究的顺利实施可为多杀菌素的异源高效合成奠定基础，并且相关的工具和研究策略可应用于其他微生物药物的高产。

多杀菌素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。.本项目在模式链霉菌Streptomyces albus J1074中异源表达了多杀菌素基因簇（~90 kb），比较了转入大片段不同遗传操作方法的有效性，实现了异源生产多杀菌素。开发了异源宿主S. albus J1074的转录组，蛋白组以及代谢组多组学检测和分析方法，锁定了异源宿主中生产多杀菌素的限速步骤，通过靶向代谢改造将异源宿主中多杀菌素产量提升1000倍，达到1 mg/L以上。为了进一步提升异源宿主中的多杀菌素产量，本项目系统研究了聚酮合酶基因在异源宿主中高表达对产物产量的影响，最终按照原始操纵子结构对spnABC三个聚酮合酶基因进行高表达和将spnD和spnE分别进行高表达的组合获得最高产菌株，产量约为30 mg/L。在此基础上，将合成聚酮合酶前体的基因进行高表达进一步提升产量至50 mg/L。随后，本项目探究了发酵培养基中残糖与多杀菌素产量的关系，证实了残糖的含量跟多杀菌素产量密切相关，并比较了5种不同的糖作为碳源，最终得到以麦芽糖为碳源的发酵培养基有助于菌株发酵生产多杀菌素，产量提升至70 mg/L。最后，本项目还测试了异源发酵的多杀菌素的杀虫活性，证实其仍然具有杀虫活性。并搭建了第一个白色链霉菌的基因组规模代谢网络模型，该模型将有助于后续继续的代谢改造工作。.本研究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。