Discovery and Biosynthesis of Bacterial Terpenoids

项目概要/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 (NPs)，（ii）阐明这些NP的生物合成途径和调节机制，以及（iii）表征 并将所发现的NP及其生物合成酶用于生物医学和生物技术应用。 纳米粒是高度功能化和进化优化的小分子，具有无与伦比的化学特性， 和结构上的差异，导致了广泛的生物活性。萜类化合物，最大和最 结构多样的NP家族被认为在细菌中很少见;只有约1.2%的已知萜类化合物是 细菌起源然而，基因组学研究表明，负责萜类化合物的生物合成酶 生物合成的蛋白质广泛分布于细菌，特别是放线菌中。我们假设（i）细菌 萜类化合物在目前的NP文库中被大大低估， 新的萜类化合物将导致新的药物线索和（ii）了解序列结构功能 萜类化合物生物合成酶的关系将为基因组挖掘、组合研究带来新的机会 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， 迫切的需求，并将为萜类化合物发现领域的持续成功奠定基础， 生物合成在第一个方向，我们将使用整合的基因组学-代谢组学方法来发现 来自细菌的新细菌萜类化合物。这将包括开发新的和创新的 用于靶向鉴定复杂细菌萜类化合物和激活或上调 萜类生物合成基因簇。在第二个方向，我们将阐明的生物合成途径， 新的和已知的细菌萜类化合物，并在功能上、机制上和结构上表征 萜烯脱氢酶及其相关氧化酶，特别是细胞色素P450。我们将使用一个 严格的多学科方法，包括基因组挖掘、生物信息学分析、体内途径 工程、（非）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，我们非常适合 在这一领域建立并维持一个成功的独立方案。此外，我们还建立了几个 与合成生物学、NP药物发现和X射线领域的领导者进行了重要合作 晶体学，进一步加强了这项研究计划。本研究计划的预期成果 包括揭示细菌萜烯组，了解萜烯如何 脱氢酶决定萜烯环化，并利用自然进化的氧化酶来产生一种新的化合物。 生物催化剂的工具箱

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.