Directed Evolution of Isoprenoid Biosynthesis

类异戊二烯生物合成的定向进化

• 批准号: 10454236
• 负责人: Gavin J Williams
• 金额: $ 29.77万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10454236
• 关键词: Address; Alcohol dependence; Anabolism; Biochemical; Biological; Biosensor; Biosynthetic Chemistry; Carbon; Chemicals; Complex; Coupled; Coupling; Cyclization; Cytochrome P450; Data; Detection; Development; Dimethylallyltranstransferase; Diphosphates; Directed Molecular Evolution; Engineering; Enzymes; Fluorescence; Goals; Health; In Situ; In Vitro; Isoprene; Lead; Medicine; Methods; Molecular; Monoterpenes; Natural Products; Nature; Organic Synthesis; Outcome; Pathway interactions; Pharmaceutical Chemistry; Pharmacology; Play; Production; Property; Public Health; Reagent; Research; Role; Route; Specificity; Structure; System; Terpenes; Therapeutic; Time; anti-cancer; flexibility; genetic manipulation; high throughput screening; human disease; in vivo; innovation; isopentenyl pyrophosphate; isoprenoid; microbial; novel; perilla alcohol; polymerization; prenyl; prototype; scaffold; small molecule therapeutics; synthetic biology; tool

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.

项目概要 异戊二烯单元是在超过 80,000 种天然产物中发现的结构基序，通常对于生物至关重要 活性和药理特性的调节。然而，我们获取类异戊二烯并使其多样化的能力 结构极其有限。类异戊二烯生物合成途径的复杂性、合成的难度 合理地克服了多个关键瓶颈，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。最后， 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 重新编程天然产物的生物合成以合成潜在的治疗药物，总体 该提案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 通过与下游生物合成偶联，形成类异戊二烯结构单元的依赖半萜 (ADH) 途径 化学并将定向进化应用于类异戊二烯生物合成。我们的假设是（1）简单性 ADH 途径有利于获得类异戊二烯，(2) 可以通过以下方式克服装饰萜烯的瓶颈 定向进化，(3)异戊烯基转移酶的有限特异性可以通过定向进化来扩展。 这些假设得到 (1) 数据的支持，这些数据验证了 ADH 途径支持生产的能力 类异戊二烯的研究，26 (2) 开发用于原位萜烯检测的基因编码生物传感器，(3) 初步 证明在体外和体内酶促生成非异戊二烯结构单元的可行性的数据 体内并将它们耦合到下游类异戊二烯生物合成，27 (4) 初步数据揭示了 异戊二烯转移酶混杂作为定向进化的平台，以及（5）异戊二烯转移酶的开发 高通量筛选。拟议研究的基本原理是我们的定向进化方法 能够获得各种医学相关的类异戊二烯，包括新的天然化合物。致地址 这些假设，并为了完成本提案的总体目标，将实现以下具体目标 已完成：(1) 通过生物传感器引导工程克服含氧萜烯的瓶颈，以及 (2) 扩展 类异戊二烯化学多样性通过异戊二烯基转移酶定向进化。我们的方法具有高度创新性，因为 类异戊二烯生物合成的定向进化以前仅限于比色萜烯和我们的化学 类异戊二烯的酶促策略提供了前所未有的范围、多功能性、模块化和实用性。拟议的 研究意义重大，因为预计它将对天然产物生物合成和 合成生物学，通过推进天然产物生物合成的新策略并实现 不易通过基因操作或传统有机方法获得的具有生物活性的天然产物 合成。