核糖体肽类天然产物细菌素的无细胞生物合成研究

Bacteriocins are ribosomally synthesized natural peptides which exhibit magnificent antimicrobial activities against many pathogens and some drug-resistant clinical pathogens. Due to they are stable and non-toxic to cells, bacteriocins potential to be developed as therapeutic alternatives to antibiotics. Although more and more bacteriocins are discovered, the study and application of bacteriocins are still limited by their low efficiency of production. In recent years, cell-free protein synthesis (CFPS) technology has been developed as a new platform for protein production due to the in vitro CFPS platforms have many advantages over in vivo microbial systems, for example, fast reaction rates, high protein yields, and tolerance of toxic products. In this project, several representative bacteriocins (Lacticin Q, Lactococcin G, Lacticin 481 and Nisin) will be synthesized by using the E. coli-based CFPS system. This study will not only pave a new avenue for the production of bacteriocins, but also provide new insights for the discovery, biosynthesis and application of ribosomally synthesized natural products. This study will further promote the next-generation biomanufacturing with high convenience, efficiency and sustainability.

核糖体肽类天然产物细菌素对多种病原菌及临床耐药菌有显著抑制作用，由于其性状稳定，且无细胞毒性，因而具有临床抗生素替代物开发的良好前景。尽管新的高活性的细菌素逐渐被发现，当前细菌素的制备存在效率低、成本高等问题，这在一定程度上限制了其更广泛的研究和应用。近年来，迅速发展的无细胞蛋白合成技术具有操作简便、反应速率快、蛋白产量高、无细胞毒害作用等诸多优势，成为蛋白表达新平台。本项目将基于大肠杆菌无细胞蛋白合成体系，选取代表性细菌素（Lacticin Q，Lactococcin G，Lacticin 481和Nisin），进行细菌素无细胞生物合成研究。该研究不仅可以提供细菌素制备的新方法，提高细菌素的制备效率，也将为基于无细胞生物合成技术进行核糖体肽类活性天然产物的开发、研究和应用奠定理论和实验基础，并进一步促进便捷、高效、节能、绿色无污染的生物合成新时代的到来。

核糖体肽类天然产物对多种病原菌及临床耐药菌有显著抑制作用，由于其性状稳定，且无细胞毒性，因而具有临床抗生素替代物开发的良好前景。目前核糖体肽类化合物的研究手段限于体内发酵和异源表达，存在步骤繁琐、效率低、成本高等问题，难以适应近年来新型核糖体肽类快速、高通量发掘研究的需求。迅速发展的无细胞蛋白合成技术具有操作简便、反应速率快、蛋白产量高、无细胞毒害作用等诸多优势，成为蛋白表达新平台。本项目基于大肠杆菌无细胞蛋白合成技术，开发构建了核糖体肽类天然产物的无细胞生物合成平台，实现了从简单核糖体肽（Lactococcin G，Cecropin P1）到复杂的翻译后修饰核糖体肽（羊毛硫肽Salivaricin B）的活性合成，并进一步研究了salivaricin B生物合成过程中修饰酶的底物泛杂性，最后筛选发掘了具有高生物活性的salivaricin B类似物。本项目的研究结果不仅提供了快速高效的核糖体肽类化合物的制备方法，也为核糖体肽类活性天然产物的生物合成、机制研究、挖掘探索和应用研究奠定理论和实验基础，并进一步促进便捷、高效、节能、绿色无污染的生物合成新时代的到来。