Liu Ran, Zhang Yuchen, Zhai Guoqing, Fu Shuai, Xia Yao, Hu Ben, Cai Xuan, Zhang Yan, Li Yan, Deng Zixin, Liu Tiangang
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education and Wuhan University School of Pharmaceutical Sciences Wuhan 430071 China.
Department of Clinical Laboratory Renmin Hospital of Wuhan University Wuhan 430060 China.
Adv Sci (Weinh). 2020 Jul 21;7(17):2001616. doi: 10.1002/advs.202001616. eCollection 2020 Sep.
Lanthipeptides have extensive therapeutic and industrial applications. However, because many are bactericidal, traditional in vivo platforms are limited in their capacity to discover and mass produce novel lanthipeptides as bacterial organisms are often critical components in these systems. Herein, the development of a cell-free protein synthesis (CFPS) platform that enables rapid genome mining, screening, and guided overproduction of lanthipeptides in vivo is described. For proof-of-concept studies, a type I lanthipeptide, nisin, is selected. Four novel lanthipeptides with antibacterial activity are identified among all nisin analogs in the National Center for Biotechnology Information (NCBI) database in a single day. Further, the CFPS platform is coupled with a screening assay for anti-gram-negative bacteria growth, resulting in the identification of a potent nisin mutant, M5. The titers of nisin and the nisin analog are found to be improved with CFPS platform guidance. Owing to the similarities in biosynthesis, the CFPS platform is broadly applicable to other lanthipeptides, thereby providing a universal method for lanthipeptide discovery and overproduction.
羊毛硫肽具有广泛的治疗和工业应用。然而，由于许多羊毛硫肽具有杀菌作用，传统的体内平台在发现和大规模生产新型羊毛硫肽方面能力有限，因为细菌生物体通常是这些系统的关键组成部分。在此，描述了一种无细胞蛋白质合成（CFPS）平台的开发，该平台能够在体内实现羊毛硫肽的快速基因组挖掘、筛选和定向过量生产。为了进行概念验证研究，选择了一种I型羊毛硫肽——乳链菌肽。一天之内就在美国国立生物技术信息中心（NCBI）数据库中的所有乳链菌肽类似物中鉴定出了四种具有抗菌活性的新型羊毛硫肽。此外，CFPS平台与抗革兰氏阴性菌生长的筛选测定相结合，从而鉴定出一种有效的乳链菌肽突变体M5。发现通过CFPS平台指导，乳链菌肽和乳链菌肽类似物的效价得到了提高。由于生物合成的相似性，CFPS平台广泛适用于其他羊毛硫肽，从而为羊毛硫肽的发现和过量生产提供了一种通用方法。
