合成生物学提倡的自下而上构建的理念以及“设计-构建-修正”的工程化策略给予天然产物研究领域两个核心问题（如何将现有重要化合物高产以及如何高效发现新的化合物）提供了新的研究手段和新的研究思路。无细胞蛋白合成体系由于解除了细胞生长与产物合成的偶联，便于进行模型设计，是合成生物学中非常重要的一个研究平台和研究手段。本申请利用无细胞蛋白合成体系对于核糖体肽类的天然产物生物合成途径在体外进行重构，以其中生物合成机理研究比较透彻的羊毛硫肽Nisin作为一个模式研究系统。首先利用无细胞体系重构Nisin的转录翻译过程以及其后修饰加工的多个生化步骤，找到这个复杂生物合成的限速步骤，并指导工业生产菌株的产量提升。然后，利用无细胞蛋白体系配上Nisin的后修乳饰系统，来筛选并合成NCBI数据库中与Nisin骨架类似的候选多肽。最后，这个系统还可以用于候选多肽的定向进化，可以通过高通量的合成和筛选获得目的多肽。

There are two major research directions in the natural product research, and one is how to produce the target molecule efficiently, the second is how to find the more and more new molecules efficiently. The concept of build the biosystem from the bottom to up and the strategy of ‘design-build-modify’ for the synthetic biology provide the new research tools and new mind to the natural product research community. Cell-free protein synthesis (CFPS) system is one of very important research platforms and methods in the synthetic biology, because CFPS is easy for preparation and it decouple the product production and host growth, CFPS is an ideal system for system design and test. . In this application, we plan to in vitro reconstituted nisin biosynthesis machinery, a famous and represented lanthipeptide, as cell-free system combining its transcription, translation, and post-translational modifications together. CFPS of nisin system will be as a model system of lanthipeptide synthesis which belongs to ribosomal synthesized post-translational modified peptides (RiPPs). We will find the rate-limiting step for biosynthesis of Nisin. After the detail investigation by using CFPS nisin system, firstly, the knowledge gained from this CFPS system will guide us to rationally engineer nisin industrial producer by targeted overexpression of key component. Secondly, all the similar peptide with nisin from NCBI sequenced database will be fast synthesized and screening of activities by using modified Nisin based CFPS system. Third, combining this cell-free system with high-throughput automation screening system, we can mutate the targeted lanthipeptide to select the desired activity. Due to the similarity between lanthipeptides, our findings and system could also be applied to engineer lanthipeptides or other ribosomal peptide in principle. This application will be an important attempt for using synthetic biology tools and concept for promotion of natural product overproduction and discovery.