Discovery and Biosynthesis of Bacterial Terpenoids

项目摘要/摘要 我们研究计划的长期目标是：(I)发现新的细菌天然产物 (Ii)阐明这些NPs的生物合成途径和调控机制，以及(Iii)表征 并将发现的NPs及其生物合成酶用于生物医学和生物技术应用。 核糖核酸是高度官能化和进化优化的小分子，拥有无与伦比的化学物质。 和结构的多样性，导致了广泛的生物活动。萜类化合物，最大和最多的 结构多样化的NPs家族，在细菌中被认为是罕见的；在已知的萜类化合物中，只有1.2%是 细菌来源。然而，基因组学研究表明，负责萜类化合物的生物合成酶 生物合成广泛存在于细菌中，尤其是放线菌中。我们假设(I)细菌 在目前的NP文库及其发现和表征中，萜类化合物被严重低估 新的萜类化合物将导致新的药物先导和(Ii)了解序列-结构-功能 萜类生物合成酶的关系将在基因组挖掘中带来新的机会，组合 生物合成和氧化生物催化。我们最初的努力遵循两个研究方向，即 并将为在萜类化合物发现和开发领域继续取得成功奠定基础 生物合成。在第一个方向，我们将使用基因组学-代谢组学的综合方法来发现 从细菌中提取的新型细菌萜类化合物。这将包括开发新的和创新的 复杂细菌萜类化合物的靶向鉴定和激活或上调的方法 萜类生物合成基因簇。在第二个方向，我们将阐明生物合成途径 新的和已知的细菌萜类化合物以及在功能、机械和结构上的表征 萜烯合成酶及其相关的氧化酶，特别是细胞色素P450。我们将使用 涉及基因组挖掘、生物信息学分析、体内途径的严格多学科方法 工程学，天然产物分离和结构测定，体外酶学，和蛋白质X-射线 结晶学。我们在萜类化合物生物合成和酶学方面的经验以及我们在 这两个研究方向都支持拟议研究的可行性，并且我们非常适合 在这一领域建立并维持一个成功的独立项目。此外，我们还建立了几个 与合成生物学、NP药物发现和X射线领域的领先者进行关键合作 进一步加强这一研究计划的结晶学。本研究计划的预期结果 包括揭示细菌萜烯组，了解萜烯如何产生的基本原理 合成酶决定了萜类的环化作用，以及利用自然进化的氧化酶来创造一种 生物催化学家的工具箱。

PROJECT SUMMARY/ABSTRACT The long-term objectives of our research program are to (i) discover novel bacterial natural products (NPs), (ii) elucidate the biosynthetic pathways and regulatory mechanisms of these NPs, and (iii) characterize and utilize the discovered NPs and their biosynthetic enzymes for biomedical and biotechnological applications. NPs are highly functionalized and evolutionarily optimized small molecules that possess unrivaled chemical and structural diversities, resulting in a wide range of biological activities. Terpenoids, the largest and most structurally diverse family of NPs, are considered rare in bacteria; only ~1.2% of known terpenoids are of bacterial origin. However, genomics studies revealed that the biosynthetic enzymes responsible for terpenoid biosynthesis are widely distributed in bacteria, particularly actinobacteria. We hypothesize that (i) bacterial terpenoids are considerably underestimated among current NP libraries and the discovery and characterization of novel terpenoids will lead to new drug leads and (ii) understanding the sequence-structure-function relationships of terpenoid biosynthetic enzymes will lead to new opportunities in genome mining, combinatorial biosynthesis, and oxidative biocatalysis. Our initial efforts follow two research directions that address immediate needs and will set the stage for continued success in the field of terpenoid discovery and biosynthesis. In the first direction, we will use an integrated genomics–metabolomics approach to discovery novel bacterial terpenoids from bacteria. This will include the development of new and innovative methodologies for targeted identification of complex bacterial terpenoids and the activation or upregulation of terpenoid biosynthetic gene clusters. In the second direction, we will elucidate the biosynthetic pathways of both new and known bacterial terpenoids and functionally, mechanistically, and structurally characterize terpene synthases and their associated oxidative enzymes, particularly cytochrome P450s. We will use a rigorous multidisciplinary approach involving genome mining, bioinformatics analysis, in vivo pathway engineering, (un)natural product isolation and structural determination, in vitro enzymology, and protein X-ray crystallography. Our experience in terpenoid biosynthesis and enzymology and our significant progress in both research directions supports the feasibility of the proposed research and that we are well-suited to establish and sustain a successful independent program in this field. In addition, we have established several key collaborations with leaders in the fields of synthetic biology, NP drug discovery, and X-ray crystallography that further strengthen this research program. Expected outcomes of this research program include the revelation of the bacterial terpenome, understanding the underlying principles of how terpene synthases dictate terpene cyclization, and the exploitation of naturally evolved oxidative enzymes to create a toolbox of biocatalysts.