Supporting data for 'Genome Mining of Uncharted Ribosomally Synthesized and Post-translationally Modified Peptides'

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32220 prospective rSAM-catalyzed RiPP BGCs from 161954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteineglycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.P450-catalyzed RiPPs stand out as a unique but underexplored RiPP family. Here, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence, co-conservation, and interactions between precursors and P450s, successfully facilitating the identification of diverse P450 catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can respectively catalyze Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s in expanding the chemical diversity of strained macrocyclic peptides and enriching biocatalytic tools for peptide macrocyclization.

核糖体合成和翻译后修饰的肽（RiPPs）代表了细菌中最大但主要未被充分探索的自然产物家族之一。RiPPs的遗传编码性质简化了其生物合成基因簇（BGCs）的预测和优先级排序。本研究报告了一种小肽与酶共现分析工作流程（SPECO），该流程使我们能够从161954个细菌基因组中识别出32220个潜在的rSAM催化的RiPP BGCs，并优先考虑了25个具有新型生物合成结构或前体模式的家族。我们鉴定了三种新的酶，分别催化半胱氨酸甘氨酸（BlaB）、组氨酸脂肪族侧链（ScaB）和酪氨酸/组氨酸精氨酸（VguB）交联。形成环烷的酶ScaB显示出广泛的底物选择性，使其能够催化多种三肽的形成。这些结果证明了SPECO工作流程在发现新酶用于肽大环化中的优势。P450催化的RiPPs作为一个独特但未被充分探索的RiPP家族脱颖而出。在此，我们介绍了一种基于规则的基因组挖掘策略，该策略利用了RiPPs固有的生物合成原理，包括前体与P450之间的共现、共保守性和相互作用，成功地促进了多种P450催化的RiPPs的识别。密集的BGC特征分析揭示了四种新的P450酶，KstB、ScnB、MciB和SgrB，它们分别能够在三个或四个残基内催化色氨酸-色氨酸-酪氨酸（一个C-C键和两个C-N键）、酪氨酸-色氨酸（C-C键）、色氨酸-色氨酸（C-N键）和组氨酸-组氨酸（醚键）交联。KstB、ScnB和MciB可以接受非天然前体，这表明它们可能是构建大环的生物工程的有前途的起始模板。本研究突出了P450在扩展应力大环肽的化学多样性和丰富肽大环化生物催化工具的潜力。