A Modular Approach for Combinatorial Biosynthesis of Functionalized Terpenoids

描述(申请人提供)：萜类化合物是最大的一类天然产物，表现出惊人的结构复杂性和相应的广泛生物活性。虽然有一些这样的化合物已经进入临床使用，但据报道，更多的化合物显示出良好的药理活性。不幸的是，进一步的调查往往受到可用材料数量的严格限制。这不仅阻碍了直接使用，也阻碍了已被证明在产生临床相关化合物方面非常有用的半合成衍生法。因此，迫切需要提供获得萜类天然产物的一般途径，但随之而来的结构复杂性阻碍了普遍适用的合成路线。作为我们设计用于药物研究和使用的目标文库和特定的单个萜类“天然”产品生产的长期目标的下一步，我们建议在我们在细菌二萜生产的模块化代谢工程方面取得的成功的基础上，通过扩展这种模块化方法来包括将萜烯类中间体精化为具有生物活性的萜类天然产品所需的多个下游氧化反应。这些氧化反应通常是由微粒体细胞色素P450与相关的还原酶结合进行的，需要广泛的工程来使这种将萜类化合物“下游”精制成萜类化合物的过程成为可能。基于我们的初步结果表明，萜烯类中间体可以有效地从为其生产而设计的细菌转移到共培养的用于后续功能化的细菌中，我们开发了一种广泛的模块化方法来设计高度功能化的萜烯类化合物的生产。这种模块化工程系统的发展将使组合生物合成的研究变得容易，许多微生物体P450表现出的底物混杂表明了组合生物合成的潜力，以及新型生物合成酶的功能鉴定。因此，除了开发拟议的模块化方法外，鉴于我们对二萜代谢的特别关注，我们将使用该代谢工程系统来对来自天然来源的二萜生物合成酶和旨在增加底物混杂的拟议酶工程努力进行功能表征。我们预计，拟议的普遍适用的模块化代谢工程系统的建立将极大地增加获得极大类别的萜类天然产物的机会。 与公共健康相关：该项目建议开发一种通用的方法，使基因工程细菌从大量的萜类天然产物(已知为50,000)中生产复杂的化合物。由此产生的小分子是潜在的药剂，其研究和使用受到相关本地生产有机体通常提供的非常有限的数量的阻碍。细菌生产将为大量生产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潜在生物活性所必需的。

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.