Mechanisms and Evolution of Assembly-Line Polyketide Synthases

装配线聚酮化合物合成酶的机制和演变

• 批准号: 10394371
• 负责人: CHAITAN KHOSLA
• 金额: $ 40.12万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10394371
• 关键词: Acyl Carrier Protein; Anabolism; Antibiotics; Biological; Biological Assay; Candidate Disease Gene; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryoelectron Microscopy; Engineering; Enzymatic Biochemistry; Enzymes; Epitopes; Evolution; Experimental Models; Gene Conversion; Genes; Genetic; Genetic Models; Goals; Human Cell Line; In Vitro; Individual; Investigation; Knock-out; Laboratories; Logic; Molecular Conformation; Monoclonal Antibodies; Names; Natural Products; Nocardia; Nocardia Infections; Nucleotides; Orphan; Phenotype; Play; Reaction; Research; Roentgen Rays; Role; Specificity; Streptomyces; Structure; Testing; base; flexibility; genetic element; genome-wide; insight; knock-down; macrophage; novel; polyketide synthase; reconstitution; small hairpin RNA; vector

PROJECT DESCRIPTION Assembly-line polyketide synthases (PKSs) are enzyme machines that catalyze vectorial biosynthesis of a growing polyketide chain through a uniquely defined sequence of acyl carrier protein and ketosynthase domains involving alternating chain translocation and elongation reactions. Notwithstanding the discovery of >3000 naturally occurring assembly-line PKSs, we do not understand how they blend catalytic specificity with evolutionary flexibility. Our lab is motivated by the goal of understanding the enzymology and evolution of assembly-line PKSs while enhancing our ability to engineer known PKSs and decode “orphan” ones. Our Goals for the next five years are to: 1) Understand the chemical logic of vectorial biosynthesis by an assembly-line PKS: We will study: (i) the structural dynamics of individual PKS modules; (ii) how different conformations of a module enable its elementary reactions; and (iii) the extent to which transitions between successive reactions are coordinated across an assembly line. Our proposed mechanistic investigations will exploit: (i) our ability to functionally reconstitute PKSs in vitro; (ii) epitope-specific monoclonal antibodies to trap individual PKS modules in specific conformational or catalytic states; and (iii) advances in X-ray, SAXS, and cryoEM analysis of PKSs. 2) De-orphanize the nocardiosis-associated NOCAP synthase: We have de-orphanized the nonamodular NOCAP synthase found in isolates of Nocardia associated with nocardiosis. Now, we propose to solve the structure of the fully tailored natural product, and to elucidate its biological role in nocardiosis. This will require us to: (i) characterize a putatively doubly glycosylated polyketide product; (ii) establish a phenotypic assay for its bioactivity in a macrophage-like human cell line; and (ii) harness genome-wide CRISPR knockout and shRNA knockdown screens to gain insight into its mode of action. 3) Decipher the role of GRINS in the evolution of assembly-line PKSs: We have discovered a new genetic element, named GRINS (genetic repeats of intense nucleotide skews), that is widespread in assembly-line PKS genes. We hypothesize that GRINS play a major role in diversifying assembly-line PKSs. To test this hypothesis, we will: (i) identify candidate genes in Streptomyces that are involved in introducing nucleotide skews or enabling gene conversion; (ii) identify a bacterial host in which gene conversion is enabled by GRINS under laboratory conditions; and (iii) develop an experimental model for GRINS-based PKS engineering. The significance of our proposal is two-fold. On one hand, it offers the opportunity to break new ground in our understanding of the structure, mechanism, and evolution of assembly-line PKSs. On the other hand, it tests the extent to which our understanding of these remarkable megasynthases can be harnessed to discover novel bioactive polyketides from “orphan” assembly-line PKSs.

项目说明 流水线聚酮合成酶(PKS)是一种催化载体生物合成的酶机器。 通过唯一定义的酰基载体蛋白和酮合成酶序列生长聚酮链 涉及交替的链移位和延伸反应的结构域。尽管发现了 &gt；3000自然产生的装配线PKS，我们不知道它们是如何将催化专一性与 进化的灵活性。我们实验室的目的是为了了解酶的作用和进化 在提高我们设计已知的PKS和破译“孤立”PKS的能力的同时，还可以提供装配线上的PKSS。 我们今后五年的奋斗目标是： 1)了解装配线上载体生物合成的化学逻辑：我们将研究：(I) 各个PKS模块的结构动力学；(Ii)一个模块的不同构象如何使其 基元反应；以及(Iii)连续反应之间的过渡被协调的程度 穿过一条装配线。我们提议的机械性调查将利用：(I)我们在功能上的能力 体外重建PKS；(Ii)表位特异性单抗捕获单个PKS模块 具体的构象或催化状态；以及(Iii)PKS的X射线、SAXS和低温电子显微镜分析的进展。 2)使诺卡氏菌病相关的NOCAP合成酶去孤儿：我们已经使非阿莫代性 与诺卡氏菌病有关的诺卡氏菌分离株中发现的NOCAP合酶。现在，我们建议解决 完全定制的天然产物的结构，并阐明其在诺卡氏菌病中的生物学作用。这将需要 美国：(I)鉴定推测的双糖化聚酮产品；(Ii)建立表型分析 其在巨噬细胞样人细胞系中的生物活性；以及(Ii)利用全基因组CRISPR基因敲除和 ShRNA敲除筛选，以深入了解其作用模式。 3)破译GRINS在装配线PKS进化中的作用：我们发现了一种新的基因 元素，称为GRINS(强烈核苷酸偏斜的遗传重复)，广泛存在于装配线上 PKS基因。我们假设，GRINS在使装配线PKS多样化方面发挥了重要作用。为了测试这一点 假设，我们将：(I)确定链霉菌中与引入核苷酸有关的候选基因 倾斜或启动基因转换；(Ii)识别通过GRINS实现基因转换的细菌宿主 以及(Iii)开发基于GRINS的PKS工程实验模型。 我们建议的意义是双重的。一方面，它提供了在我们的 了解装配线PKSS的结构、机制和发展。另一方面，它测试了 我们对这些非凡的超级合成酶的了解可以在多大程度上被利用来发现 来自“孤儿”流水线PKS的新型生物活性多酮。