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

一种能够重新编程非核糖体肽酶学的进化方法

• 批准号: 1716594
• 负责人: Michael Thomas
• 金额: $ 75万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1716594&HistoricalAwards=false
• 关键词: evolutionary; approach; enable; reprogramming; non

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.

非核糖体肽是由许多细菌自然产生的，并且是疗法的极好来源，最著名的是新的抗生素。 许多这些肽的合成涉及一类称为非核糖体肽合酶的酶。 这些酶以类似于带有酶工作站或“模块”协调构建肽一个氨基酸的酶的装配线的方式起作用。 该项目的目的是学习从不同非核糖体肽合酶中混合和匹配工作站的规则。 最终，这将使研究人员能够构造合成新肽的杂化酶，然后筛选肽的治疗特性。 由于生物科学和工程科学的教师之间的合作，这项研究将提供跨学科培训。 这种跨学科的方法将纳入正式的本科盖石课程，一项本科夏季研究计划，并向K-12学生推广。 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. 凝结（C）域随后以方向性方式形成两个相邻的PCP系氨基酸之间的酰胺键，形成肽主链。 结构和生化研究已经确定了定义域识别的氨基酸的域特异性代码。 尽管该代码在预测被激活的氨基酸方面非常有用，但许多研究表明，改变定义该代码的残基几乎总是无法切换底物特异性。 此外，完整的域或模块掉期生成嵌合非核糖体肽合成酶通常无法有效地发挥作用，这可能是由于蛋白质 - 蛋白质相互作用不当或相关C域的底物特异性所致。该项目将使用体内定向的进化方法研究非核糖体肽合成酶的A和C结构域的重编程。 使用这些方法，本研究旨在定义一种用于生成功能性嵌合A域的方法，确定新的A域特异性代码，以改变氨基酸识别，并了解C域管理底物识别的残基。 这些研究的结果将有助于我们合理地重新编程这种重要类别天然产品的生物合成，以生成设计器分子以出于各种应用目的，同时也可以深入了解自然如何实现这一目标。

项目成果

Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code

定向进化揭示腺苷酸化域特异性代码的功能序列空间

• DOI: 10.1021/acschembio.9b00532
• 发表时间: 2019
• 期刊: ACS Chemical Biology
• 影响因子: 4
• 作者: Throckmorton, Kurt;Vinnik, Vladimir;Chowdhury, Ratul;Cook, Taylor;Chevrette, Marc G.;Maranas, Costas;Pfleger, Brian;Thomas, Michael George
• 通讯作者: Thomas, Michael George

Leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous hosts

• DOI: 10.1039/c9md00055k
• 发表时间: 2019-05-01
• 期刊: MEDCHEMCOMM
• 作者: Cook, Taylor B.;Pfleger, Brian F.
• 通讯作者: Pfleger, Brian F.

Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.

• DOI: 10.1016/j.ymben.2021.06.004
• 发表时间: 2021-09
• 期刊: Metabolic engineering
• 影响因子: 8.4
• 作者: Cook TB;Jacobson TB;Venkataraman MV;Hofstetter H;Amador-Noguez D;Thomas MG;Pfleger BF
• 通讯作者: Pfleger BF