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: 10582364
• 负责人: Shanteri Singh
• 金额: $ 14.06万
• 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/10582364
• 关键词: Acids; Active Sites; Address; Alcohols; Award; Cannabinoids; Cells; Collaborations; Coupled; Coupling; Diphosphates; Engineering; Environment; Enzymes; Escherichia coli; Farnesol; Funding; 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; Parents; 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

Summary of the funded parent award (1R01GM138800-01A1). 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.

资助父母奖摘要(1R01GM138800-01A1)。类异戊二烯代表了一类不同的 在医药和工业中有广泛应用的化合物。它们是从自然来源中提取出来的 既对环境具有挑战性，又对环境有潜在的危害，而许多 异戊二烯类化合物使传统的化学合成变得非常重要。现代代谢工程与合成生物学 方法已经克服了其中的一些困难，但与代谢流量和有限的 通用的类异戊二烯前体的可获得性使其广泛实施变得复杂。人造的 到目前为止，所开发的方法仅集中于合成二甲基烯丙基和异戊烯基二磷酸酯 并需要额外的酶来产生聚戊烯基二磷酸盐(聚戊烯基聚苯硫醚)。因此， 这项建议的主要目标是开发一种有效的策略来合成天然和非天然的 用于下游应用的(聚)戊烯基聚苯硫醚。这将使用两种相辅相成的方法来实现：一) 使用十一碳烯醇激动剂和异戊烯基磷酸激动剂；以及ii)使用羟乙基噻唑 激酶、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结合，在聚戊烯基中加入额外的多样性- PPS。拟议的研究包括：一)选定酶的结构和功能评估；二)催化剂 工程学，以及iii)体外和体内耦合平台的优化，以产生多样化的 精选天然产品的资料库。我们预计这些研究将产生：i)规则和概念得到推进 了解特定类别的酶的构效关系；ii)优化的酶偶联 产生多样化底物和类异戊二烯的平台；以及iii)具有潜力的新型类异戊二烯类似物 治疗应用。因此，这项拟议的工作将提供前所未有的途径，获得独特的生物活性 不容易通过传统方法访问的类异戊二烯文库，它将深化我们的基本 了解四种酶的种类，同时将它们发展成有用的生物催化剂。