High throughput biosynthesis of ribosomally synthesized and post-translationally modified peptide natural products

核糖体合成和翻译后修饰肽天然产物的高通量生物合成

• 批准号: 10417229
• 负责人: Mark Chalfant Walker
• 金额: $ 34.33万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10417229
• 关键词: Anabolism; Antibiotics; Antineoplastic Agents; Biological; Chemicals; Diagnosis; Disease; Dwarfism; Enzymes; Fluorescence-Activated Cell Sorting; Focus Groups; Gene Library; Health; Human; Libraries; Mutation Analysis; Natural Products; Nature; Organism; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Ribosomes; Surface; Testing; Variant; Yeasts; base; enzyme substrate; genome sequencing; next generation sequencing; novel strategies; small molecule; tool

Chemicals produced by living systems, or natural products, have had a tremendous impact on human health. For example, nearly one third of all small molecule drugs approved by a regulatory agency over the past nearly four decades have been natural products or derivatives of natural products, including over 70% of antibiotics and 40% of anticancer drugs [1]. While very useful molecules have been identified among the hundreds of thousands of natural products that have been characterized to date, genome sequencing efforts over the past decade and a half have revealed that we have only characterized the products of a small fraction of the biosynthetic pathways that exist in nature. The products of these pathways have the potential to greatly impact the diagnosis and treatment of disease, and it is critical that we develop new approaches to accelerate the identification and characterization of new natural products and natural product-like compounds. Towards this critical need, my group focuses on the ribosomally synthesized and post-translationally modified peptide (RiPP) class of natural products [2], and the enzymes involved in their biosynthesis. Unique among natural product biosynthetic pathways, the substrate of the RiPP biosynthetic enzymes is a genetically encoded precursor peptide. This feature of the substrates allows for deep mutational analysis, not just of the enzymes, but of their substrates as well. We will develop a platform for high throughput examination of the substrate selectivity and activity of RiPP biosynthetic enzymes based on yeast or bacterial surface display, fluorescence activated cell sorting, and next generation sequencing. Using this platform and gene libraries encoding substrate variants we will study the substrate scope of these enzymes, and how that scope relates to the sequence of the native substrate of the enzymes. Additionally, we will perform deep mutational analysis of the enzymes to identify contributions to substrate recognition and selectivity. These studies will provide us with a deeper understanding of how these enzymes function. With this deeper understanding, we will be able to use these enzymes as tools to generate large libraries of natural product-like compounds that can be screened to identify those with useful biological activities more efficiently than current natural product discovery approaches.

生命系统产生的化学物质或天然产品对人类健康产生了巨大的影响。 例如，过去监管机构批准的所有小分子药物中，近三分之一几乎 四十年来一直是天然产品或天然产品的衍生物，包括70%以上的抗生素和 40%的抗癌药物[1]。虽然已经在数十万个分子中发现了非常有用的分子 到目前为止已经被表征的天然产品，过去十年的基因组测序工作以及 一半的人透露，我们只对一小部分生物合成途径的产物进行了表征。 它们存在于自然界中。这些途径的产物有可能极大地影响诊断和 疾病的治疗，至关重要的是我们开发新的方法来加速识别和 新的天然产物和天然产物类化合物的表征。 针对这一关键需求，我的团队专注于核糖体合成和翻译后修饰 多肽(RIPP)类天然产物[2]，以及参与其生物合成的酶。独一无二的 天然产物生物合成途径中，RIPP生物合成酶的底物是一种遗传编码的 前体多肽。底物的这一特征允许进行深入的突变分析，不仅是酶的分析， 也包括它们的底物。我们将开发一个基片高通量检测平台 基于酵母或细菌表面展示、荧光的RIPP生物合成酶的选择性和活性 激活了细胞分类和下一代测序。利用该平台和编码底物的基因文库 我们将研究这些酶的底物范围，以及该范围与 酶的天然底物。此外，我们将对这些酶进行深入的突变分析，以确定 对底物识别和选择性的贡献。这些研究将为我们提供更深入的理解 这些酶是如何发挥作用的。有了这个更深入的了解，我们将能够将这些酶用作工具 生成大量的天然产物类化合物，可以对这些化合物进行筛选以识别那些有用的化合物 生物活动比目前的天然产品发现方法更有效。