Accurate Broadcasting of Substrate Fitness for Lactazole Biosynthetic Pathway from Reactivity-Profiling mRNA Display
Accurate Broadcasting of Substrate Fitness for Lactazole Biosynthetic Pathway from Reactivity-Profiling mRNA Display
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从反应性分析 mRNA 显示准确广播乳唑生物合成途径的底物适应性
DOI：
10.1021/jacs.0c10374
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发表时间：
2020
影响因子：
15
通讯作者：
Suga Hiroaki
中科院分区：
文献类型：
--
作者：
Vinogradov Alexander A.;Nagai Emiko;Chang Jun Shi;Narumi Kakeru;Onaka Hiroyasu;Goto Yuki;Suga Hiroaki
我们报告了一种方法的高通量反应性分析的遗传编码库作为一种工具，研究底物健身景观RiPP（核糖体合成和后修饰肽）生物合成酶。这种方法使我们能够快速分析的底物偏好的lactazole生物合成途径使用饱和诱变mRNA展示库的lactazole前体肽。我们证明，该测定法产生准确和可重复的体外数据，使定量的反应产量与时间分辨率。我们的研究结果概括了以前建立的知识lactazole的生物合成和扩大它通过确定的程度表现出的酶底物混杂。本工作为构建和筛选基于mRNA展示的组合硫肽文库，发现具有全新生物活性的乳唑硫肽奠定了基础。
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.