Enzyme-Mediated Synthesis of Functionalized Terpene Structures

项目摘要/摘要本申请涉及广泛的挑战领域（06），使能技术和特定的挑战主题06- gm -109：药物发现、开发和生产的绿色化学和工程。萜烯是由生物系统合成的最多样化的化合物之一，已被用作抗菌、抗真菌和抗癌剂，用于治疗人类疾病。尽管许多萜烯作为治疗靶点具有很高的价值，但由于萜烯核心存在几个手性中心，并且需要对官能团进行不同的保护和去保护，化学合成路线已被证明是难以捉摸的。在过去的十年中，利用金属基催化剂进行区域和立体特异性反应的复杂功能化萜烯的化学合成取得了巨大的进展，并且获得少量的萜烯已经足以确认一些候选药物的治疗价值。然而，未来我们需要具有成本效益和绿色的合成方法来生产足够数量的治疗性萜烯，以用于药物开发和商业化。为了解决这一问题，我们提出了一个绿色化学平台来合成具有治疗价值的萜烯烯烃和功能化萜烯。首先，通过在工程微生物宿主中表达选定的已知和新型萜烯合成酶，我们将生成构成治疗化合物核心结构的萜烯烯烃。其次，我们将使用高效细菌细胞色素P450单加氧酶的突变体来功能化这些萜烯烯烃。最后，我们将优化这些酶在微生物菌株中的表达，我们已经设计了过量生产通用萜烯前体，以实现高水平生产具有治疗价值的萜烯烯烃和氧化萜烯。该方案的成功完成将为经济、清洁地合成具有治疗价值的萜烯烯烃和氧化萜烯提供一个微生物平台。这项工作对人类健康有三个重大影响。首先，微生物萜烯可以用作合成完整的治疗性萜烯和萜烯类似物的后期中间体，从而加速发现基于萜烯的治疗方法。其次，与依赖昂贵的起始材料和有毒溶剂和催化剂的传统有机合成方法相比，微生物生产萜烯更具成本效益和环境友好性。最后，该平台的通用性将通过在宿主菌株中引入和诱变新酶来简化其他功能化萜烯的合成。

DESCRIPTION (provided by applicant): Enzyme-Mediated Synthesis of Functionalized Terpene Structures Project Summary/Abstract This application addresses broad challenge area (06), Enabling Technologies, and specific Challenge Topic 06-GM-109: Green chemistry and engineering for drug discovery, development, and production. Terpenes, constituting one of the most diverse groups of compounds synthesized by biological systems, have been used as antibacterial, antifungal, and anticancer agents in the treatment of human disease. Despite the high value of many terpenes as therapeutic targets, chemical synthesis routes have proven elusive due to the presence of several chiral centers in the terpene core and the need for differential protection and deprotection of functional groups. In the last ten years there has been tremendous progress in the chemical synthesis of complex functionalized terpenes using metal-based catalysts to execute regio and stereospecific reactions, and having access to small quantities of terpenes has been sufficient to confirm the therapeutic value of a number of drug candidates. However, going forward we will need cost-effective and green synthetic methods to produce quantities of therapeutic terpenes sufficient for drug development and commercialization. To address this problem, we propose a green chemistry platform for the synthesis of terpene olefins and functionalized terpenes of therapeutic value. First, through expression of selected known and novel terpene synthases in engineered microbial hosts, we will generate terpene olefins that constitute core structures of therapeutic compounds. Second, we will functionalize these terpene olefins using mutants of a highly efficient bacterial cytochrome P450 monooxygenase. Finally, we will optimize expression of these enzymes in microbial strains that we have engineered to overproduce universal terpene precursors in order to achieve high-level production of terpene olefins and oxidized terpenes of therapeutic value. Successful completion of this proposal will provide a microbial platform for the economical and clean synthesis of terpene olefins and oxidized terpenes of therapeutic value. This work has three significant impacts on human health. First, the microbial terpenes can then be used as late intermediates in the synthesis of complete therapeutic terpenes and terpene analogs, thus accelerating the discovery of terpene-based therapeutics. Second, microbial production of terpenes is more cost-effective and environment-friendly when compared to traditional organic synthesis methods that rely on expensive starting materials and toxic solvents and catalysts. Finally, the generality of the proposed platform will streamline the synthesis of other functionalized terpenes via the introduction and mutagenesis of new enzymes in our host strains. PUBLIC HEALTH RELEVANCE: There are many natural products in the terpene family with promising or proven therapeutic characteristics. As yields from natural sources are frequently low, and typical chemical synthesis routes are often challenging and expensive, we aim to facilitate economical and clean enzyme-mediated synthesis of a number of these products. In addition, our plan enables green synthesis of product analogs for drug discovery and improvement programs though the use of mutant enzymes.