Directed Evolution of Isoprenoid Biosynthesis

类异戊二烯生物合成的定向进化

• 批准号: 10280273
• 负责人: Gavin J Williams
• 金额: $ 29.47万
• 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/10280273
• 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.

项目摘要 异戊二烯单元是在> 80,000天然产品中发现的结构基序，通常对于生物学至关重要 药理特性的活性和调节。然而，我们获得类异型素的能力并使他们的多样化 结构非常有限。类异丙生素生物合成途径的复杂性，难度 理性地克服多个关键瓶颈，许多组件的狭窄底物范围具有 妨碍了前进工程类异丙因生物合成以扩大化学空间的尝试。最后， 异普尼生物合成的全合成潜力尚未实现。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剂，这是整体 该建议的目的是利用我们最近描述的酒精的简单性，模块化和多功能性 依赖的半萜（ADH）途径通过将其耦合到下游生物合成来通往类异型构建块 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） ADH途径有助于使用类异丙素，（2）可以克服装饰萜烯的瓶颈 定向进化，（3）可以通过定向进化来扩展前转移酶的有限特异性。 这些假设得到了（1）数据，该数据验证了ADH途径支持生产的能力 类异on-，26（2）开发用于原位萜烯检测的遗传编码的生物传感器，（3）初步 数据表明，酶上生成非异戊二烯构建块的可行性，并在 体内并将它们耦合到下游的类异型生物合成，27（4）揭示的初步数据 前转移酶滥交是定向进化的平台，以及（5）前转移酶的发展 高通量屏幕。拟议研究的理由是我们定向进化的方法 启用了各种与药物相关的类化合物的访问，包括新的化合物。解决 这些假设，并完成该提案的总体目标，以下具体目标将是 完成：（1）通过生物传感器引导的工程克服瓶颈到氧化的萜烯，（2）扩展 类化学多样性通过前转移酶定向进化。我们的方法是高度创新的，因为 异普尼生物合成的定向进化以前仅限于比色萜烯和我们的化学 类异丙因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。提议 研究之所以重要，是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自然产品生物合成的新策略并使能够访问 生物活性天然产品不容易通过遗传操作或常规有机物访问 合成。