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物对人类健康产生了巨大影响。 例如，过去监管机构批准的所有小分子药物中有近三分之一 四十年来都是天然产物或天然产物的衍生物，其中70%以上的抗生素和 抗癌药物占40%[1]。虽然已经在数十万种分子中发现了非常有用的分子 迄今为止已表征的天然产物、过去十年的基因组测序工作以及 一半的人透露，我们只表征了一小部分生物合成途径的产物 存在于自然界中的。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方法来加速识别和治疗 新天然产物和类天然产物化合物的表征。 为了满足这一迫切需求，我的团队专注于核糖体合成和翻译后修饰 肽（RiPP）类天然产物[2]，以及参与其生物合成的酶。独一无二 天然产物生物合成途径，RiPP生物合成酶的底物是基因编码的 前体肽。底物的这一特征允许进行深度突变分析，而不仅仅是酶的突变分析， 但也包括它们的底物。我们将开发一个用于基板高通量检查的平台 基于酵母或细菌表面显示、荧光的 RiPP 生物合成酶的选择性和活性 激活细胞分选和下一代测序。使用该平台和编码底物的基因库 我们将研究这些酶的底物范围，以及该范围与酶序列的关系 酶的天然底物。此外，我们将对酶进行深度突变分析，以确定 对底物识别和选择性的贡献。这些研究将使我们有更深入的了解 这些酶如何发挥作用。有了更深入的了解，我们将能够使用这些酶作为工具 生成类似天然产物的化合物的大型库，可以对其进行筛选以识别那些有用的化合物 生物活性比当前的天然产物发现方法更有效。