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.

项目描述 组装线聚酮化合物合酶（PKS）是促催化近代生物合成的酶 通过独特定义的酰基载体蛋白和酮合酶的序列生长的聚酮链链 涉及替代链易位和伸长反应的域。尽管发现 > 3000个天然存在的装配线PKS，我们不了解它们如何将催化特异性与 进化灵活性。我们的实验室的动机是理解酶学和演变的目标 组装行PKS同时增强了我们设计已知PKS并解码“孤儿”的能力。 我们接下来五年的目标是： 1）通过组装行PKS了解矢量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）模块的不同考虑如何使其能够 基本反应； （iii）成功反应之间的过渡的程度 跨组装线。我们提出的机械投资将利用：（i）我们在功能上的能力 在体外重构PKS； （ii）表位特异性单克隆抗体可将单个PKS模块捕获 特定会议或催化状态； （iii）PKS的X射线，SAXS和冷冻分析的进步。 2）将甲型甲氧化作用相关的NOCAP合酶：我们已脱苯甲酸化 NOCAP合酶在与Nocardiosis相关的Nocardia分离株中发现。现在，我们建议解决 完全量身定制的天然产物的结构，并阐明其生物学在诺卡症中的作用。这将需要 我们要：（i）表征推定的双倍糖基化聚酮化合物； （ii）建立表型测定 它在巨噬细胞状的人类细胞系中的生物活性； （ii）宽松基因组CRISPR敲除和 shRNA敲低筛选以深入了解其作用方式。 3）破译笑容在组装线PKS演变中的作用：我们发现了一个新的通用 元素，名为Grins（强烈的核苷酸偏斜的遗传重复），在组装线上很普遍 PKS基因。我们假设笑容在多样化的组装线PKS中起着重要作用。测试这个 假设，我们将：（i）确定参与引入核苷酸的链霉菌中的候选基因 偏向或启用基因转换； （ii）确定一个细菌宿主，在该宿主中启用基因转换 在实验室条件下； （iii）为基于笑的PKS工程开发了一个实验模型。 我们的提议的意义是两个方面。一方面，它提供了机会在我们的 了解组装线PKS的结构，机制和演变。另一方面，它测试 我们对这些非凡的巨型统计的理解的程度可以利用 来自“孤儿”组装线PKS的新型生物活性聚酮化合物。