结合组学技术进行安丝菌素生产的发酵工程研究

Microorganisms produce numerous valuable secondary metabolites, and their fermentation processes are complicated. In-depth studies on the fermentation processes of microbial secondary metabolites have significant theoretical and applied values. This project will take the production of an important secondary metabolite - antitumor ansamitocin by Actinosynnema pretiosum as a research target, by combining with omics (transcriptomics, proteomics and metabolomics) analysis. The affecting mechanism of the effects of important environmental factors on ansamitocin production wil be elucidated. An engineered strain of Actinosynnema pretiosum (producing ansamitocin) will be bred by deleting negative regulatory genes and enhancing the expressiont of positive regulatory genes for ansamitocin biosynthesis. The fermentation performance of the engineered strain will be evaluated in fermentors for potenatil large-scale application. The comparative analysis of metabolic network, metabolic flux spectrum, protein expression and their relationship with key environmental factors for wild-type and engineered strains will be conducted. Then, the engineered strain will be further modified for higher production. Scale-up of the fermentation process from 2 liter to 100 liter fermentor will be done, and the scale-up rule will be found out. Based on the information obtained, new ideas and new methodologies for highly efficient production of the valuable secondary metabolite will be proposed. The study will help to deepen our understanding of the regulation mechanism of the secondary metabolite biosynthesis as well as to reach large-scale efficient production. The research approaches, ideas and methods will be beneficial to in-depth study on fermentation engineering for production of other secondary metabolites.

微生物生产众多有用次级代谢产物，其发酵过程较为复杂。针对微生物次级代谢发酵过程的深入研究具有重要的理论和应用价值。本课题以珍贵橙色束丝放线菌生产一种重要次级代谢物- - 抗肿瘤安丝菌素作为研究对象，结合组学（包括基因转录谱、蛋白质谱、代谢物谱）分析，阐明重要环境因子对其发酵过程影响的机理。构建缺失负调节基因、增强正调节基因表达的安丝菌素高产的工程菌，并在发酵罐中发酵验证。通过对比分析野生菌和工程菌的主要代谢网络和代谢流量谱、蛋白质表达及与重要环境因子的关系，完善工程菌的构建，并实现从2立升到100立升发酵罐的过程放大，获得放大规律。基于获得的信息，提出安丝菌素高效发酵生产的新思路新方法。该研究有助于加深了解该抗癌药物生物合成的调节机制及其大规模高效生产，有关研究方法和思路也有益于其他次级代谢物发酵生产的深入研究。

微生物生产众多有用次级代谢产物，其发酵过程复杂。无疑，针对微生物次级代谢发酵过程的深入研究具有重要的理论和应用价值。本课题以珍贵橙色束丝放线菌生产一种重要次级代谢物- - 抗肿瘤安丝菌素作为研究对象，在国家自然科学基金和上海市优秀学科带头人计划的先后资助下，在生产菌株突变筛选、代谢工程理性改造、发酵过程调控及放大，取得了重要进展。结合组学分析，阐明重要环境因子对其发酵过程影响的机理。构建了缺失负调节基因、增强前体生物合成基因表达的安丝菌素高产的工程菌4株，并在发酵罐中发酵验证。对比分析了野生菌和工程菌的主要代谢网络和代谢流量谱、基因转录及与重要环境因子的关系。实现了从摇瓶到实验室小规模发酵罐、直至125立升发酵罐的过程放大，获得放大规律。基于获得的信息，提出安丝菌素高效发酵生产的新思路新方法。该研究有助于加深了解该抗癌药物生物合成的调节机制及其大规模高效生产，有关研究方法和思路也有益于其他次级代谢物发酵生产的深入研究。部分成果已以科学论文的形式发表在生物工程领域顶尖期刊《Biotechnology and Bioengineering》以及《Trends in Biotechnology》，受邀在国内外学术会议做大会报告和邀请报告，并受邀编著出版国际学术专著2本。共发表相关期刊和学术会议等论著11篇；有关提高安丝菌素生物合成产量方法的研究成果已申请发明专利1项。后续相关研究尚待进一步完善发表。