Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

中文翻译：

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无细胞的生物合成，以评估套索肽的形成和酶底物的耐受性。

套索肽是核糖体合成的和翻译后修饰的肽（RiPP）天然产物，具有独特的套索状结构。由于具有严格的拓扑结构，套索肽对热和蛋白水解降解异常稳定。已显示某些套索肽结合人细胞表面受体并表现出抗癌特性，而另一些则表现出抗菌或抗病毒活性。已知的套索肽是由细菌产生的，基因组挖掘研究表明，套索肽是相对普遍的RiPP类。然而，缺乏有效的生产系统限制了套索肽的发现，分离和表征。在这项研究中，我们采用无细胞生物合成（CFB）策略来解决与套索肽生产相关的长期挑战。我们报告成功地形成了套索多肽的多样化阵列，其中包括已知的例子，以及来自嗜盐嗜热菌的新的预测套索肽。我们进一步证明了CFB可以快速生成和表征多位点前体肽变体，以评估生物合成途径的底物耐受性。我们显示，来自fusilassin途径的套索形成环化酶可以通过CFB产生数百万个序列多样的套索肽，在套索肽的环区域内具有非同寻常的水平序列容许性。这些数据为使用CFB识别具有独特特性的新变体创建大型套索肽库奠定了坚实的基础。