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.

项目概要 类异戊二烯代表了多种化合物，在医学和工业中具有广泛的应用。 从自然资源中提取它们既具有挑战性，又对环境有潜在危害，而 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非常重要。现代的 代谢工程和合成生物学方法已经克服了其中一些困难，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其复杂化 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酯，需要额外的酶来生成聚异戊二烯基- 二磷酸酯（聚异戊二烯-PP）。因此，本提案的主要目标是制定有效的战略 用于下游应用的天然和非天然（聚）异戊二烯基聚丙烯的合成。这将实现 使用两种互补的方法：i)使用十一烯醇激酶和异戊烯磷酸激酶；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结合使用 聚异戊二烯-PP 的额外多样性。拟议的研究包括： i) 结构和功能评估 选定的酶，ii) 催化剂工程，以及 iii) 耦合体外和体内平台的优化 生成精选天然产品的多样化库。我们期望这些研究能够产生： i) 规则和 增进对选定类别酶的结构-活性关系的了解的概念； ii) 优化的， 产生多样化底物和类异戊二烯的酶偶联平台； iii) 新型类异戊二烯类似物 具有潜在的治疗应用。因此，拟议的工作将提供前所未有的独特途径 生物活性类异戊二烯库无法通过传统方法轻易获得，并且它将加深我们的研究 对四种酶的基本了解，同时将它们开发成有用的生物催化剂。