Directed Evolution of Isoprenoid Biosynthesis

项目摘要 异戊二烯单元是在> 80，000种天然产物中发现的结构基序，并且通常对生物活性至关重要。 活性和药理学性质的调节。然而，我们获取类异戊二烯并使其多样化的能力， 结构非常有限。类异戊二烯生物合成途径的复杂性， 合理克服多个关键瓶颈，许多元器件衬底范围狭窄， 阻碍了对类异戊二烯生物合成进行正向工程化以扩大化学空间的尝试。因此，委员会认为， 类异戊二烯生物合成的全部合成潜力还有待实现。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， 这个提议的目的是利用我们最近描述的酒精的简单性、模块性和多功能性 通过将依赖性半萜（ADH）途径与下游生物合成 化学和应用定向进化类异戊二烯生物合成。我们的假设是（1）简单的 ADH途径有助于获得类异戊二烯，（2）修饰萜烯的瓶颈可以通过以下方式克服： 定向进化，和（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。 这些假设得到以下方面的支持：（1）验证ADH途径支持生产能力的数据 （2）开发用于原位萜烯检测的遗传编码生物传感器，（3）初步 这些数据证明了在体外和体内酶促产生非异戊二烯结构单元的可行性， 体内并将其与下游类异戊二烯生物合成偶联，27（4）初步数据显示， 异戊烯基转移酶混杂作为定向进化的平台，以及（5）异戊烯基转移酶的开发 高通量筛选。这项研究的基本原理是，我们的定向进化方法 使得能够获得各种医学上相关的类异戊二烯，包括新的天然化合物。解决 这些假设，并完成这一建议的总体目标，以下具体目标将是 完成：（1）通过生物传感器引导工程克服含氧萜烯的瓶颈，（2）扩大 通过异戊烯基转移酶定向进化的类异戊二烯化学多样性。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烯，而我们的化学方法， 类异戊二烯的酶促策略提供了前所未有的范围、通用性、模块性和实用性。拟议 研究是重要的，因为它预计在天然产物生物合成中具有广泛的积极影响， 合成生物学通过推进天然产物生物合成的新策略， 生物活性天然产品不容易通过遗传操作或常规有机 合成.

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.