Discovery of novel ribosomally synthesized and post-translationally modified peptides (RiPPs) originating from unusual two-domain precursors

次级代谢产物（天然产物）代表了自然界的“进化的化学游乐场”，因为这些化合物显示出特殊的结构多样性。通常，这些代谢物具有有效的活性，其中许多被用于制药应用。因此，新的次级代谢产物的鉴定和相应的生物合成酶的表征对于理解进化这种巨大化学多样性的遗传和生化机制具有重要意义。此外，具有生物活性的新型化合物的发现使得能够开发新的抗感染药，鉴于抗生素耐药性的增加，迫切需要这种抗感染药。该项目的重点是一组独特的核糖体合成和后修饰的肽（RIPPs）从细菌。RiPP是一大类次级代谢产物，其衍生自经历翻译后修饰的遗传编码的前体肽。前体肽通常由前导序列和核心序列组成。不同的酶识别前导序列并在核心肽上进行翻译后修饰，核心肽在蛋白水解切割后作为成熟的天然产物释放。前导序列通常很短，缺乏明确的结构特征。作为例外，“腈水合酶前导肽”（NHLP）家族的特征在于显示出与腈水合酶相似性的异常长的前导序列。在伯克霍尔德菌目的某些物种中，这些NHLP前体进一步表现为串联基因，即，前体的两个拷贝存在于一行中。有趣的是，在一些菌株中，这些基因融合成一个单一的双结构域前体，产生一个大的~270个氨基酸的前体蛋白。这两个前导结构域的功能以及所产生的RIPP的性质目前尚不清楚。这些前体的独特结构域结构可能影响所得肽的成熟，并代表了发现具有新结构和功能的RIPP的未开发和令人兴奋的机会。在这个项目中，我们的目标是研究这些迄今尚未表征的RiPP基因簇，以确定产生的代谢产物，表征生物合成酶，并探索两个前导结构域在前体中的作用。我们将采用自上而下的方法，包括代谢组学和蛋白质组学，以确定和分离的RIPPs从生产生物体。我们将进一步补充这些方法与自下而上的方法，我们将迭代重建大肠杆菌中的生物合成基因，以监测前体中修饰的连续分期付款。为了了解前体中两个NHLP结构域的功能，我们计划将蛋白质结晶并解析其结构。最后，将评估分离的RIPP的生物活性，以评价其抗感染潜力。

Secondary metabolites (natural products) represent nature’s “chemical playground of evolution”, as these compounds show an exceptional structural diversity. Often, these metabolites have potent activities and many of them are harnessed for pharmaceutical applications. The identification of novel secondary metabolites and the characterization of the corresponding biosynthetic enzymes are thus of great importance to understand the genetic and biochemical mechanisms that evolved this great chemical diversity. Further, the discovery of novel compounds with biological activities enables the development of new anti-infectives, which are urgently needed in light of increased antibiotic resistance. This project focuses on a unique group of ribosomally synthesized and post-translationally modified peptides (RiPPs) from bacteria. RiPPs are a large class of secondary metabolites that are derived from genetically encoded precursor peptides that undergo post-translation modification. The precursor peptides usually consist of a leader sequence and a core sequence. Different enzymes recognize the leader and install post-translational modifications on the core peptide, which is released as the mature natural product after proteolytic cleavage. Leader sequences are often short and lack clear structural features. As an exception, the family of the “nitrile hydratase leader peptides” (NHLP) are characterized by unusually long leader sequences that show similarity to the enzyme nitrile hydratase. In certain species of the order Burkholderiales, these NHLP precursors further appear as tandem genes, i.e., two copies of the precursor are present in a row. Intriguingly, in a few strains, these genes are fused into a single two-domain precursor, resulting in a large ~270 amino acid precursor protein. The function of the two leader domains, as well as the nature of the resulting RiPPs is currently unclear. The unique domain architecture of these precursors likely affects the maturation of the resulting peptides and represents an unexplored and exciting opportunity for the discovery of RiPPs with novel structures and functions. In this project, we aim to investigate these so-far uncharacterized RiPP gene clusters to identify the produced metabolite(s), characterize the biosynthetic enzymes, and explore the role of the two leader domains in the precursors. We will employ top-down approaches, including metabolomics and proteomics, in order to identify and isolate the RiPPs from the producing organisms. We will further complement these methods with a bottom-up approach where we will iteratively reconstitute the biosynthetic genes in Escherichia coli to monitor the successive installment of modifications in the precursor. To understand the function of the two NHLP domains in the precursor, we plan to crystallize the protein and solve its structure. Lastly, isolated RiPPs will be assessed for their bioactivities to evaluate their anti-infective potential.