We report a method for the high-throughput reactivity profiling of genetically encoded libraries as a tool to study substrate fitness landscapes for RiPP (ribosomally synthesized and post-translationally modified peptide) biosynthetic enzymes. This method allowed us to rapidly analyze the substrate preferences of the lactazole biosynthetic pathway using a saturation mutagenesis mRNA display library of lactazole precursor peptides. We demonstrate that the assay produces accurate and reproducible in vitro data, enabling the quantification of reaction yields with temporal resolution. Our results recapitulate the previously established knowledge on lactazole biosynthesis and expand it by identifying the extent of substrate promiscuity exhibited by the enzymes. This work lays a foundation for the construction and screening of mRNA display-based combinatorial thiopeptide libraries for the discovery of lactazole-inspired thiopeptides with de novo designed biological activities.

中文翻译：

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从反应性分析 mRNA 显示准确广播乳酸唑生物合成途径的底物适合度

我们报告了一种对基因编码文库进行高通量反应性分析的方法，作为研究 RiPP（核糖体合成和翻译后修饰肽）生物合成酶底物适应性景观的工具。这种方法使我们能够使用乳唑前体肽的饱和诱变 mRNA 展示库快速分析乳唑生物合成途径的底物偏好。我们证明该测定可产生准确且可重复的体外数据，从而能够以时间分辨率对反应产率进行量化。我们的结果概括了先前建立的关于乳唑生物合成的知识，并通过确定酶表现出的底物混杂程度来扩展它。