School of Physical Science and Technology, ShanghaiTech University, Shanghai, China.
State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai, China.
Nat Commun. 2024 May 21;15(1):4336. doi: 10.1038/s41467-024-48726-y.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.
核糖体合成和翻译后修饰肽 (RiPPs) 是一类具有多种化学结构和强大生物活性的天然产物。大多数 RiPP 基因簇在微生物基因组中尚未被探索，这部分是由于缺乏快速有效的异源表达系统来进行 RiPP 的表征和生物合成。在这里，我们报告了一种基于无细胞基因表达的统一生物催化 (UniBioCat) 系统，用于 RiPP 的快速生物合成和工程改造。我们通过重新构建从头生物合成唾液菌素 B 的完整生物合成途径来证明 UniBioCat 的作用，唾液菌素 B 是一种 lanthipeptide RiPP。接下来，我们从源菌株中删除了几个蛋白酶/肽酶基因，以增强 UniBioCat 的性能，然后可以合成和筛选具有增强抗菌活性的唾液菌素 B 变体。最后，我们通过合成和评估十种未表征的 lanthipeptides 的生物活性来表明 UniBioCat 的通用性。我们预计 UniBioCat 将加速 RiPP 的发现、表征和合成。
