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: 10205865
• 负责人: CHAITAN KHOSLA
• 金额: $ 40.1万
• 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/10205865
• 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.

项目描述 装配线聚酮酶（PKS）是催化多聚体的载体生物合成的酶机器， 通过酰基载体蛋白和酮合酶的独特定义序列生长聚酮链 涉及交替链易位和延伸反应的结构域。尽管发现了 >3000个天然存在的装配线PKS，我们不明白它们如何将催化特异性与 进化的灵活性我们的实验室的动机是了解酶学和进化的目标， 组装线PKS，同时提高我们的能力，工程师已知的PKS和解码“孤儿”的。 我们今后五年的目标是： 1)理解装配线PKS的载体生物合成的化学逻辑：我们将研究：（i） 单个PKS模块的结构动力学;（ii）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协调的程度 穿过一条装配线我们提出的机制研究将利用：（i）我们的能力， （ii）表位特异性单克隆抗体，以捕获PKS中的单个PKS模块; 特定的构象或催化状态;和（iii）在X射线，SAXS，和冷冻电镜分析PKSs的进展。 2)使诺卡氏菌病相关的NOCAP合酶去乙酰化：我们已经使非调节性的NOCAP合酶去乙酰化。 在与诺卡氏菌病相关的诺卡氏菌分离株中发现NOCAP合酶。现在，我们建议解决 结构的完全定制的天然产品，并阐明其在诺卡氏菌病的生物学作用。这将需要 我们：（i）表征一种纯化的双糖基化聚酮产物;（ii）建立一种表型测定法， 其在巨噬细胞样人细胞系中的生物活性;和（ii）利用全基因组CRISPR敲除， shRNA敲除筛选以深入了解其作用模式。 3)解读GRINS在装配线PKS进化中的作用：我们发现了一种新的基因 一种广泛存在于装配线上的称为GRINS（强烈核苷酸偏斜的遗传重复）的元件 PKS基因。我们假设，GRINS发挥了重要作用，在多样化的装配线PKS。为了验证这一 假设，我们将：（i）鉴定链霉菌中参与引入核苷酸候选基因 （ii）鉴定其中GRINS使基因转化成为可能的细菌宿主 在实验室条件下;和（iii）开发基于GRINS的PKS工程的实验模型。 我们的建议具有双重意义。一方面，它提供了一个机会，在我们的 理解装配线PKS的结构、机制和演变。另一方面，它测试了 我们对这些非凡的巨型合成酶的理解， 来自“孤儿”装配线PKS的新型生物活性聚酮化合物。