A Chemoenzymatic Approach to Accessing Novel Isoprenoid Scaffolds

受资助的母公司奖励汇总表（1R01GM138800 - 01A1）。类异戊二烯代表了一个不同的类别， 这些化合物在医药和工业中具有广泛的应用。从自然资源中提取的 既具有挑战性，又可能对环境有害，而许多建筑的巨大结构复杂性， 类异戊二烯使传统的化学合成变得不平凡。现代代谢工程与合成生物学 方法已经克服了这些困难中的一些，但是与代谢通量相关的问题和有限的 通用类异戊二烯前体的可用性使其广泛应用复杂化。人工 迄今为止开发的途径仅集中于合成二甲基烯丙基和异戊烯基二磷酸酯 并且需要额外的酶来产生聚异戊二烯基-二磷酸（聚异戊二烯基-PP）。因此 该提案的主要目标是开发一种有效的策略，用于合成天然和非天然的 用于下游应用的（聚）异戊二烯基-PP。这将通过两种互补的方法来实现： 使用十一异戊二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，将额外的多样性引入聚异戊二烯基- PP。拟议的研究包括：i）选定酶的结构和功能评估，ii）催化剂 工程化，和iii）优化偶联的体外和体内平台，用于产生多样化的 精选天然产品的图书馆。我们希望这些研究能够产生：i）规则和概念，以促进 在选定类别的酶的结构-活性关系的知识; ii）优化的，酶偶联的 产生多样化底物和类异戊二烯的平台;和iii）具有潜在的新的类异戊二烯类似物 治疗应用。因此，拟议的工作将提供前所未有的获得独特的生物活性， 通过传统方法不易获得的类异戊二烯库，它将加深我们的基础 了解四种酶，同时将它们开发成有用的生物催化剂。

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.