A Modular Approach for Combinatorial Biosynthesis of Functionalized Terpenoids

功能化萜类化合物组合生物合成的模块化方法

• 批准号: 7693208
• 负责人: REUBEN JOHN PETERS
• 金额: $ 22万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7693208
• 关键词: Active Sites; Alkenes; Anabolism; Antibiotics; Antimalarials; Antineoplastic Agents; Applications Grants; Area; Artemisinins; Attention; Bacteria; Biological; Biological Factors; Biological Models; Biological Process; Carbon; Cells; Chemical Structure; Class; Classification; Clinical; Cloning; Coculture Techniques; Complex; Cytochrome P450; Derivation procedure; Development; Diffuse; Diterpenes; Engineering; Environment; Enzymes; Escherichia coli; Exhibits; Family; Genes; Genetic Transcription; Gibberellins; Goals; Individual; Investigation; Isoprene; Labdanes; Lead; Libraries; Medicine; Membrane; Metabolic; Metabolic Pathway; Metabolism; Methods; Microbe; Modification; Mono-S; Mycoses; Nature; Numbers; Organism; Oxidases; Oxidoreductase; Oxygen; Oxygenases; Paclitaxel; Pathway interactions; Pharmacologic Substance; Phytochemical; Plant Growth Regulators; Plants; Production; Public Health; Publishing; Range; Reaction; Recombinants; Refractory; Reporting; Research; Rice; Route; Site; Source; Specificity; Structure; Structure-Activity Relationship; System; Terpenes; Terpenoid Biosynthesis Pathway; Testing; Today; Translating; Variant; Work; analog; artemisinine; base; clinical application; clinically relevant; combinatorial; design; ent-kaurene oxidase; expectation; experience; functional group; innovation; isoprenoid; labdane; microbial; momilactone A; novel; prevent; reconstitution; response; small molecule; success

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.

描述（由申请人提供）：萜类化合物是最大的一类天然产物，具有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，更多的化合物显示出有希望的药物活性。不幸的是，进一步的调查往往受到可用材料数量的严重限制。这不仅阻碍了直接使用，而且也阻碍了半合成衍生方法，这种方法已被证明在生成临床相关化合物方面非常有用。因此，迫切需要提供萜类天然产物的一般途径，但伴随的结构复杂性阻碍了普遍适用的合成路线。下一步，我们的长期目标是为药物研究和使用而设计生产目标文库和特定的单个萜“天然”产物，我们建议通过扩展这种模块化方法，包括将萜烯烯烃中间体精制为生物活性萜天然产物所需的多种下游氧化反应，从而在细菌二萜生产的模块化代谢工程方面取得成功。这些氧化反应通常由微粒体细胞色素P450与相关的还原酶合作进行，需要大量的工程设计才能使萜烯“下游”细化为萜类。我们的初步结果表明，萜烯烯烃中间体可以有效地从用于生产的细菌转移到用于后续功能化的共培养细菌中，我们已经开发了一种广泛的模块化方法来设计高功能化萜的生产。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，其潜力是由许多微粒体p450所表现出的底物混杂性所表明的，以及新型生物合成酶的功能鉴定。因此，除了开发所提出的模块化方法，并考虑到我们对二萜代谢的特别关注，我们将使用这种代谢工程系统来功能表征来自天然来源和旨在增加底物乱交的酶工程努力的二萜生物合成酶。我们期望建立普遍适用的模块化代谢工程系统，以方便地组装扩展的生物合成途径，将极大地增加对极大类萜类天然产物的获取。