An evolutionary approach to enable reprogramming of non-ribosomal peptide enzymology

非核糖体多肽是由许多细菌自然产生的，是治疗药物的极好来源，最引人注目的是新抗生素。其中许多多肽的合成涉及一类称为非核糖体多肽合成酶的酶。这些酶的功能类似于带有酶工作站或“模块”的装配线，它们一次协调制造一个氨基酸的多肽。该项目的目标是学习混合和匹配来自不同非核糖体多肽合成酶的工作站的规则。最终，这将使研究人员能够构建合成新型多肽的杂交酶，然后筛选这些多肽的治疗特性。由于生物科学和工程科学的教师之间的合作努力，这项研究将提供跨学科的培训。这种跨学科的方法将被整合到正式的本科生顶峰课程、本科生暑期研究计划以及K-12学生的外展活动中。这种方法还将使学生接触到基本的科学问题(例如底物识别)，并向学生展示如何将这些信息应用于一个重要的目标(例如，生产设计分子)。在非核糖体多肽组装过程中，腺化(A)结构域识别特定的氨基酸，并将它们连接到伙伴多肽载体蛋白(PCP)结构域。缩合(C)结构域随后以定向方式在两个相邻的PCP系留氨基酸之间形成酰胺键，形成肽骨架。结构和生化研究已经确定了定义该结构域识别的氨基酸的A结构域专一性编码。虽然这个密码在预测激活的氨基酸方面被证明是非常有用的，但一些研究表明，改变定义这个密码的残基几乎总是无法改变底物的特异性。此外，产生嵌合型非核糖体肽合成酶的完整结构域或模块交换通常无法有效发挥作用，这可能是由于相关C结构域的不正确蛋白质相互作用或底物特异性所致。这个项目将使用体内定向进化方法来研究非核糖体多肽合成酶的A和C结构域的重新编程。利用这些方法，本研究旨在定义一种产生功能性嵌合A结构域的方法，识别新的改变氨基酸识别的A结构域专一性编码，并了解C结构域控制底物识别的残基。这些研究的结果将有助于我们对这类重要天然产品的生物合成进行合理的重新编程，以产生用于各种应用目的的设计分子，同时还可以深入了解大自然是如何实现这一目标的。

Non-ribosomal peptides are naturally produced by many bacteria and have been an excellent source of therapeutics, most notably new antibiotics. The synthesis of many of these peptides involves a class of enzymes called non-ribosomal peptide synthetases. These enzymes function in a manner analogous to an assembly line with enzyme workstations or 'modules' that coordinately build the peptide one amino acid at a time. The goal of this project is to learn the rules for mixing and matching the workstations from different non-ribosomal peptide synthetases. Ultimately, this would allow researchers to construct hybrid enzymes that synthesize novel peptides and then screen the peptides for therapeutic properties. This research will provide cross-disciplinary training due to the collaborative effort between faculty in the biological sciences and engineering sciences. This cross-disciplinary approach will be integrated into a formal undergraduate capstone course, an undergraduate summer research program, and outreach to K-12 students. This approach will also expose the students to basic scientific questions (e.g. substrate recognition) and show the students how this information can be applied to an important goal (e.g. production of designer molecules).During non-ribosomal peptide assembly, the adenylation (A) domains recognize specific amino acids and tethers them to partner peptidyl carrier protein (PCP) domains. Condensation (C) domains subsequently form amide bonds between two neighboring PCP-tethered amino acids in a directional manner, forming the peptide backbone. Structural and biochemical studies have identified A domain specificity codes that define the amino acid recognized by the domain. While this code has proved enormously useful in predicting the amino acid activated, a number of studies have shown that altering the residues that define this code nearly always fail to switch substrate specificity. Furthermore, complete domain or module swaps generating chimeric non-ribosomal peptide synthetases generally fail to function efficiently, likely due to improper protein-protein interactions or substrate specificity of the associated C domains. This project will investigate the reprogramming of both A and C domains of non-ribosomal peptide synthetases using in vivo directed evolution approaches. Using these approaches, this study aims to define a means for generating functional chimeric A domains, identify new A domain specificity codes for altering amino acid recognition, and understand the residues governing substrate recognition by C domains. Results from these studies will aid our ability to rationally reprogram the biosynthesis of this important class of natural products to generate designer molecules for a variety of applied purposes, while also gaining insights into how nature has accomplished this goal.