A Chemoenzymatic Approach to Accessing Novel Isoprenoid Scaffolds

获取新型类异戊二烯支架的化学酶方法

• 批准号: 10364914
• 负责人: Shanteri Singh
• 金额: $ 30.51万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10364914
• 关键词: Acids; Active Sites; Address; Alcohols; Cannabinoids; Cells; Collaborations; Coupled; Coupling; Diphosphates; Engineering; Environment; Enzymes; Escherichia coli; Farnesol; Generations; Geranyltranstransferase; Goals; Human; In Situ; In Vitro; Industry; Isoprene; Kinetics; Knowledge; Libraries; Medicine; Metabolic; Metabolic Pathway; Methionine; Methodology; Methods; Methyltransferase; Minnesota; Modernization; Monitor; Mutagenesis; Natural Products; Nature; North Carolina; Pathway interactions; Phosphotransferases; Process; Production; Reaction; S-Adenosylmethionine; Source; Squalene; Squalene Synthetase; Structure; Structure-Activity Relationship; System; Technology; Therapeutic; Universities; Variant; Work; analog; analytical method; base; catalyst; chemical synthesis; combinatorial; farnesyl pyrophosphate; high throughput screening; in vivo; inorganic phosphate; isopentenyl pyrophosphate; isoprenoid; metabolic engineering; methionine adenosyltransferase; non-Native; novel; prenyl; scaffold; synthetic biology

Project Summary Isoprenoids represent a diverse class of compounds with a broad range of applications in medicine and industry. Their extraction from natural sources is both challenging and potentially harmful to the environment, while the enormous structural complexity of many isoprenoids makes traditional chemical synthesis nontrivial. Modern metabolic engineering and synthetic biology approaches have overcome some of these difficulties, but issues related to metabolic flux and the limited availability of the universal isoprenoid precursors complicate their widespread implementation. The artificial pathways developed thus far have been solely focused on synthesizing dimethylallyl and isopentenyl diphosphates and require additional enzymes for the generation of polyprenyl- diphosphates (polyprenyl-PPs). Thus, the primary objective of this proposal is to develop an efficient strategy for the synthesis of both natural and unnatural (poly)prenyl-PPs for downstream applications. This will be achieved using two complementary methods: i) employing undecaprenol kinases and isopentenyl phosphate kinases; and ii) employing hydroxyethylthiazole kinase, isopentenyl phosphate kinases, and farnesyl diphosphate synthase. Additionally, the two methods will work in conjunction with isoprenoid methylatransferases to incorporate additional diversity into the polyprenyl-PPs. The proposed studies include: i) structural and functional assessment of selected enzymes, ii) catalyst engineering, and iii) optimization of coupled in vitro and in vivo platforms for the generation of diversified libraries of select natural products. We expect these studies to generate: i) rules and concepts to advance knowledge on structure-activity relationships in selected classes of enzymes; ii) an optimized, enzyme-coupled platform to generate diversified substrates and isoprenoids; and iii) novel isoprenoid analogs with potential therapeutic applications. Thus, the proposed work will offer unprecedented access to uniquely bioactive isoprenoid libraries not readily accessible via traditional methods, and it stands to deepen our fundamental understanding of four enzyme classes while also developing them into useful biocatalysts.

项目摘要 异戊二烯类化合物是一类在医药和工业中有着广泛应用的化合物。 从自然来源中提取它们既具有挑战性，又对环境有潜在的危害，而 许多异戊二烯类化合物的巨大结构复杂性使传统的化学合成变得非常重要。现代 代谢工程和合成生物学方法克服了其中的一些困难，但问题是 与代谢流量和通用类异戊二烯前体的有限可获得性有关的问题使其复杂化 广泛实施。到目前为止，开发的人工通路仅专注于合成 二甲基烯丙基和异戊烯基二磷酸盐，需要额外的酶来产生聚戊烯基二磷酸盐- 二磷酸盐(聚戊烯基聚苯硫醚)。因此，这项提议的主要目标是制定一项有效的战略 用于下游应用的天然和非天然(聚)戊烯基聚苯硫醚的合成。这将会实现的 使用两种相辅相成的方法：i)使用十一碳烯醇激动酶和异戊烯基磷酸激动酶；以及 Ii)使用羟乙基噻唑酶、异戊烯基磷酸酶和法尼基二磷酸合成酶。 此外，这两种方法将与类异戊二烯甲基转移酶一起工作，以结合 聚戊烯基聚苯硫醚的额外多样性。拟议的研究包括：一)结构和功能评估 选定的酶，ii)催化剂工程，以及iii)体外和体内耦合平台的优化 精选天然产品的多样化图书馆的产生。我们预计这些研究将产生：i)规则和 概念，以促进关于选定类别的酶的结构-活性关系的知识；ii)优化的， 用于产生各种底物和类异戊二烯的酶偶联平台；以及iii)新的类异戊二烯类似物 具有潜在的治疗应用。因此，拟议的工作将提供前所未有的访问独特的 生物活性异戊二烯文库不容易通过传统方法访问，它将深化我们的 对四种酶的基本了解，同时也将它们发展成有用的生物催化剂。