Directed Evolution of Isoprenoid Biosynthesis

项目总结 异戊二烯单元是一种结构基序，存在于80,000种天然产品中，通常对生物 药理性质的活性和调节。然而，我们获得异戊二烯并使其多样化的能力 结构一直非常有限。类异戊二烯生物合成途径的复杂性，以及 合理地克服多个关键瓶颈，以及许多组件的狭窄基板范围 阻碍了对类异戊二烯生物合成进行正向工程以扩大化学空间的尝试。因此， 类异戊二烯生物合成的全部合成潜力尚未实现。作为长期目标的一部分， 对天然产物的生物合成进行重新编程，以合成潜在的治疗药物 这项建议的目标是利用我们最近描述的酒精的简单性、模块化和多功能性 依赖半萜(ADH)途径通过偶联下游生物合成获得类异戊二烯积木 化学和定向进化在类异戊二烯生物合成中的应用。我们的假设是(1)简单性 ADH途径有助于获得异戊二烯类化合物，(2)修饰萜类化合物的瓶颈可以通过 定向进化，以及(3)戊烯基转移酶的有限专一性可以通过定向进化来扩大。 这些假设得到了(1)证实ADH途径支持生产的能力的数据的支持 对于异戊二烯，26(2)用于原位检测萜类化合物的遗传编码生物传感器的开发，(3)初步 证明酶法在体外和体外生成非异戊二烯构建块可行性的数据 并将它们与下游类异戊二烯的生物合成偶联，27(4)初步数据显示 作为定向进化平台的异戊基转移酶混杂，以及(5)异戊烯基转移酶的发展 高通量屏幕。提出这项研究的基本原理是，我们的定向进化方法 能够获得各种医学上相关的异戊二烯类化合物，包括新的自然化合物。致信地址 这些假设，以及为了完成这项提案的总体目标，将有以下具体目标 完成：(1)通过生物传感器引导的工程攻克含氧萜类化合物的瓶颈；(2)扩大 异戊二烯类化学多样性通过异戊二烯转移酶引导的进化。我们的方法是高度创新的，因为 类异戊二烯生物合成的定向进化以前仅限于比色萜类化合物和我们的化学和化学合成。 酶促类异戊二烯策略提供了前所未有的范围、多功能性、模块化和实用性。建议数 这项研究意义重大，因为预计它将对天然产物的生物合成和 通过推进天然产物生物合成的新战略并使人们能够获得 具有生物活性的天然产物，不易通过基因操作或传统有机方式获得 综合。

PROJECT SUMMARY The isoprene unit is a structural motif found in >80,000 natural products and is usually critical for biological activity and modulation of pharmacological properties. Yet, our ability to access isoprenoids and diversify their structures has been extremely limited. The complexity of isoprenoid biosynthetic pathways, the difficulty of rationally overcoming multiple critical bottlenecks, and the narrow substrate scope of many components have hampered attempts at forward engineering isoprenoid biosynthesis to expand chemical space. Consequently, the full synthetic potential of isoprenoid biosynthesis has yet to be realized. As part of the long-term goal of reprogramming the biosynthesis of natural products for the synthesis of potential therapeutics, the overall objective of this proposal is to leverage the simplicity, modularity, and versatility of our recently described Alcohol Dependent Hemiterpene (ADH) pathway to isoprenoid building blocks by coupling it to downstream biosynthetic chemistry and applying directed evolution to isoprenoid biosynthesis. Our hypotheses are (1) the simplicity of the ADH pathway facilitates access to isoprenoids, (2) bottlenecks to decorated terpenes can be overcome by directed evolution, and (3) the limited specificity of prenyltransferases can be expanded by directed evolution. These hypotheses are supported by (1) data that validates the ability of the ADH pathway to support production of isoprenoids,26 (2) development of genetically-encoded biosensors for in situ terpene detection, (3) preliminary data that demonstrates the feasibility of enzymatically generating non-isoprene building blocks in vitro and in vivo and coupling them to downstream isoprenoid biosynthesis,27 (4) preliminary data that reveals prenyltransferase promiscuity as a platform for directed evolution, and (5) development of a prenyltransferase high-throughput screen. The rationale for the proposed research is that our approach of directed evolution enables access to a variety of medicinally relevant isoprenoids including new-to-nature compounds. To address these hypotheses, and to complete the overall objective of this proposal, the following specific aims will be completed: (1) overcome bottlenecks to oxygenated terpenes via biosensor-guided engineering and (2) expand isoprenoid chemical diversity via prenyltransferase directed evolution. Our approach is highly innovative because directed evolution of isoprenoid biosynthesis has previously been limited to colorimetric terpenes and our chemo- enzymatic strategy to isoprenoids offers unprecedented scope, versatility, modularity, and utility. The proposed research is significant because it is expected to have broad positive impact in natural product biosynthesis and synthetic biology by advancing new strategies for natural product biosynthesis and enabling access to biologically active natural products not readily accessible by genetic manipulation or conventional organic synthesis.