A Modular Approach for Combinatorial Biosynthesis of Functionalized Terpenoids

描述（由申请人提供）：萜类化合物形成最大类别的天然产物，表现出惊人的结构复杂性和相应的广泛的生物活性。虽然一些这样的化合物已经进入临床使用，但据报道，更多的化合物表现出有希望的药物活性。不幸的是，进一步的调查往往受到严重限制的数量可用的材料。这不仅阻碍了直接使用，而且也阻碍了半合成衍生方法，该方法已被证明在产生临床相关化合物方面非常有用。因此，迫切需要提供对萜类天然产物的一般获取，然而伴随的结构复杂性阻止了一般适用的合成路线。作为实现我们长期目标的下一步，即设计生产用于药物研究和使用的靶向文库和特定的单个萜类“天然”产品，我们建议在我们在细菌二萜生产的模块化代谢工程方面的成功基础上，通过扩展这种模块化方法，以包括将萜烯烃中间体精细加工成生物活性萜类天然产物所需的多个下游氧化反应。产品.这些氧合反应通常由微粒体细胞色素P450与相关还原酶合作进行，需要广泛的工程改造以实现萜烯到萜类化合物的这种“下游”加工。基于我们的初步结果，表明萜烯烯烃中间体可以有效地从为其生产而工程化的细菌转移到为随后的功能化而工程化的共培养细菌，我们已经开发了一种广泛的模块化方法来工程化生产高度功能化的萜类化合物。这种模块化工程系统的发展将使组合生物合成，其潜力是由许多微粒体P450表现出的底物混杂，以及新的生物合成酶的功能鉴定的建议，方便的调查。因此，除了提出的模块化方法的发展，并考虑到我们特别关注的二萜类代谢，我们将使用这个代谢工程系统的功能特性二萜类生物合成酶从天然来源和拟议的酶工程的努力，旨在增加底物混杂。我们预计，建立拟议的普遍适用的模块化代谢工程系统的扩展的生物合成途径的简易组装将显着增加获得非常大的类萜类天然产物。 公共卫生相关性：该项目提出了一种通用的方法，用于基因工程细菌从大量的萜类天然产物（已知> 50，000）中生产精细化合物。所得的小分子是潜在的药剂，其研究和使用受到通常可从相关天然生产生物体获得的非常有限的量的阻碍。细菌生产将为生产大量的这些天然产物提供有效的来源，这些天然产物是详细研究其有前途的生物活性所必需的。

DESCRIPTION (provided by applicant): Terpenoids form the largest class of natural products, exhibiting astounding structural complexity and a correspondingly wide range of biological activity. While a few such compounds have found their way into clinical use, many more have been reported to exhibit promising pharmaceutical activity. Unfortunately, further investigation is often restricted by severe limitations in amount of available material. This hinders not only direct use, but also the semi-synthetic derivation approach that has proven so useful in generating clinically relevant compounds. Thus, there is a pressing need to provide general access to terpenoid natural products, yet the accompanying structural complexity prevents generally applicable synthetic routes. As the next step towards our long-term goal of engineering the production of targeted libraries and specific individual terpenoid `natural' products for pharmaceutical investigation and use, we propose to build on our success in modular metabolic engineering of bacterial diterpene production by expanding this modular approach to include the multiple downstream oxygenation reactions required for elaboration of the terpene olefin intermediates to bioactive terpenoid natural products. These oxygenation reactions are typically carried out by microsomal cytochromes P450 in partnership with an associated reductase, requiring extensive engineering to enable such `downstream' elaboration of terpenes to terpenoids. Based on our preliminary results indicating that the terpene olefin intermediate can be efficiently transferred from bacteria engineered for its production to co-cultured bacteria engineered for subsequent functionalization, we have developed an extensively modular approach to engineering the production of highly functionalized terpenoids. Development of this modular engineering system will enable facile investigation of combinatorial biosynthesis, whose potential is suggested by the substrate promiscuity exhibited by many microsomal P450s, as well as functional identification of novel biosynthetic enzymes. Accordingly, in addition to development of the proposed modular approach, and given our particular focus on diterpenoid metabolism, we will use this metabolic engineering system to functionally characterize diterpenoid biosynthetic enzymes from both native sources and proposed enzymatic engineering efforts designed to increase substrate promiscuity. We expect that establishment of the proposed generally applicable modular metabolic engineering system for facile assembly of extended biosynthetic pathways will dramatically increase access to the extremely large class of terpenoid natural products. PUBLIC HEALTH RELEVANCE: This project proposes development of a general method for genetically engineering bacteria to produce elaborate compounds from the enormous class of terpenoid natural products (>50,000 known). The resulting small molecules are potential pharmaceutical agents whose investigation and use has been hindered by the very limited quantities that are typically available from the relevant native producing organism. Bacterial production will provide an effective source for production of the larger amounts of these natural products necessary for detailed investigation of their promising biological activity.