Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA-dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. Significance The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, e.g. of their evolution, activity and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

中文翻译：

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Streptomyces lividans 66 通过利用 tRNA 的酶与 C-NRPS 相互作用产生蛋白酶抑制剂

具有蛋白酶抑制活性的小肽醛 (SPA) 是天然存在的化合物，由非核糖体肽合成酶 (NRPS) 合成。SPA 广泛应用于生物技术并已被用作治疗剂。它们也具有生理相关性，并且被认为能够调节其生产微生物的发育。此前，我们在Streptomyces lividans 66中鉴定了一个类似NRPS的生物合成基因簇，该基因簇缺乏缩合（C）结构域，但包含属于亮氨酰/苯丙氨酰（L/F）转移酶家族的tRNA利用酶（tRUE）。该系统预计将指导新型 SPA 的合成，我们将其命名为 Livipeptin。使用进化基因组挖掘方法，我们确认了多种链霉菌和相关生物体的基因组中存在 L/F 转移酶 tRUE，包括与预期的 C-minus NRPS 样蛋白的融合。然后，我们证明了已识别的 L/F 转移酶趋异 tRUE 同源物与 C--NRPS 之间的遗传功能协作，从而导致具有蛋白酶抑制活性的代谢级分的合成。在 RNAse 存在下的半合成测定表明，tRUE 和 C-minus NRPS 酶之间的有效相互作用确实是 tRNA 依赖性的。我们希望通过利用未发现的遗传基础来合成假定的乙酰-亮氨酸/苯丙氨酸-精氨酸蛋白酶抑制剂，我们的研究结果将促进 SPA 的发现以及蛋白酶介导的生物技术的发展。此外，这些结果将有助于利维肽素的纯化和结构阐明，而利维肽素已被证明难以进行化学表征。意义 天然产物生物合成基因的发现标志着我们对这些代谢物（例如它们的进化、活性和生物合成）理解的重大进步，而且也为推进基因组挖掘方法提供了生物技术机会和知识。我们通过在浅青紫链霉菌 66 中发现了一条新的生物合成途径，该途径显示出可指导强蛋白酶抑制剂（称为 Livipeptin）的合成，并遵循前所未有的生物合成规则和基因，从而使这成为可能。因此，通过揭示预测控制乙酰亮氨酸/苯丙精氨酸蛋白酶抑制剂（包括难以捉摸的利维肽素）产生的遗传机制，这项研究使新型蛋白酶介导的生物技术以及从基因组数据中发现蛋白酶抑制剂的方法成为可能。