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: 10543814
• 负责人: Shanteri Singh
• 金额: $ 29.11万
• 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/10543814
• 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; Natural Selections; Natural Source; Nature; North Carolina; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Production; Reaction; S-Adenosylhomocysteine; S-Adenosylmethionine; Squalene; Squalene Synthetase; Structure; Structure-Activity Relationship; System; Technology; Therapeutic; Universities; Variant; Work; analog; analytical method; 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 类异戊二烯代表了在医学和工业中具有广泛应用的不同类别的化合物。 从自然资源中提取这些物质既具有挑战性，又可能对环境有害， 许多类异戊二烯的巨大结构复杂性使得传统的化学合成变得不简单。现代 代谢工程和合成生物学方法已经克服了这些困难中的一些，但问题 与代谢通量相关的和通用类异戊二烯前体的有限可用性使它们的 广泛实施。迄今为止开发的人工途径仅集中于合成 二甲基烯丙基和异戊烯基二磷酸，并需要额外的酶来产生聚异戊二烯- 二磷酸盐（聚异戊二烯基-PP）。因此，本提案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）异戊二烯基-PP的合成。完成这项工作的方法是 使用两种互补的方法：i）使用十一烯醇激酶和异戊烯基磷酸激酶;和 ii）使用羟乙基噻唑激酶、异戊烯基磷酸激酶和法呢基二磷酸合酶。 此外，这两种方法将与类异戊二烯甲基转移酶一起工作， 聚异戊二烯基-PP的额外多样性。建议的研究包括：i）结构及功能评估 ii）催化剂工程化，和iii）优化偶联的体外和体内平台， 产生多样化的精选天然产物库。我们希望这些研究能够产生：i）规则和 概念，以提高对所选酶类别的结构-活性关系的认识; ii）优化的， 产生多样化底物和类异戊二烯的酶偶联平台;和iii）新的类异戊二烯类似物 具有潜在的治疗应用。因此，拟议中的工作将提供前所未有的机会， 生物活性类异戊二烯库不容易通过传统方法获得，它将深化我们的研究。 对四种酶的基本理解，同时也将它们开发成有用的生物催化剂。