Discovery and Characterization of Natural Product Systems

 描述(由申请人提供)：这个拟议的MIRA项目采用了一系列多学科方法来发现和分析天然产品以及组装和修饰复杂代谢物的生物合成途径。该提案涵盖了NIGMS在过去20年中支持的三个领域。每一次都被表述为一项重大挑战，旨在补充我们的成就，并继续大力推进，以发现新知识并提供具有改善人类健康潜力的解决方案。这个Mira应用程序的第一大挑战是基于我的团队领导的一个高生产率和协作性计划的激动人心的势头，该计划重点关注匹克罗霉素(Pik)、红霉素(Debs)、泰乐菌素(TYL)、咖喱(Cur)和苔藓抑素(Bry)途径，其详细分析在上一个支持周期中得到了进一步的发展。每个系统都具有令人着迷的生化特征，将扩大我们对底物选择性的理解，以及结构特征，使天然和工程聚酮合成酶/非核糖体多肽合成酶模块内部和之间的功能活性得以实现。这项提案的大挑战II侧重于与天然产物途径剪裁酶相关的研究。次生代谢结构多样化的一个基本方面涉及对生物活性有重大贡献的氧化过程。这可以很容易地在一些重要的分子中被认识到，这些分子是临床治疗药物，或者显示出作为药物先导的巨大潜力。基于我们在过去四年中在P450底物和酶工程方面的研究取得的重要成就，我们有勇气向令人兴奋的新方向扩展我们的工作。这包括计划研究一系列催化迭代氧化过程的P450单加氧酶。我们还将研究在分子间和分子内氧化反应中催化C-C偶联的单加氧酶，包括芳香族、烷基和烯基官能团。一类最未被研究但却非常重要的剪裁酶包括依赖于酰基/肽基载体蛋白的单加氧酶，我们建议探索选择性的机制，并继续努力扩大它们的底物识别和生物催化性能。第三大挑战的重点是天然产物发现和路径工程。我们已经建立了技术和生物信息学能力，可以轻松地组装和挖掘来自不同微生物群体的基因组和元基因组数据集，以实现天然产物基因簇的发现，这现在已经准备好在顺从的微生物宿主中异源表达。下一波的进展将依赖于对最新途径的现成识别，以及我们使用简便的合成生物学方法表达这些途径的能力。我们计划以最大的精力和决心解决这些问题，以获得具有宝贵药用价值的重要化合物。

 DESCRIPTION (provided by applicant): This proposed MIRA project employs a range of multi-disciplinary approaches toward the discovery and analysis of natural products and the biosynthetic pathways that assemble and modify complex metabolites. The proposal covers three areas that have been supported by NIGMS during the past 20 years. Each has been articulated as a Grand Challenge designed to complement our accomplishments and continue to push forward vigorously to discover new knowledge and offer solutions with high potential for improving human health. Grand Challenge I of this MIRA application is based on the exciting momentum of a highly productive and collaborative program lead by my group that focuses on the pikromycin (Pik), erythromycin (DEBS), tylosin (Tyl), curacin (Cur) and bryostatin (Bry) pathways whose detailed analysis has been further developed during the previous cycle of support. These systems each bear fascinating biochemical features that will expand our understanding of substrate selectivity, and structural characteristics that enable functional activity within and between native and engineered polyketide synthase/non-ribosomal peptide synthetase modules. Grand Challenge II of this proposal focuses on studies relating to natural product pathway tailoring enzymes. A fundamental aspect of structural diversification in secondary metabolism involves oxidative processes that contribute significantly to biological activity. This can be readily appreciated in a number of important molecules that are clinical therapeutic agents, or show significant potential as drug leads. Based on the important successes in our research relating to P450 substrate and enzyme engineering over the past four years, we have been emboldened to expand our work in exciting new directions. This includes plans to investigate a range of P450 monoxygenases that catalyze iterative oxidative processes. We will also investigate monooxygenases that catalyze C-C coupling involving substrates in both inter- and intramolecular oxidation reactions, including aromatic, alkyl and alkenyl functional groups. One of the most underexplored, yet very important classes of tailoring enzyme includes the acyl/peptidyl carrier protein dependent monooxygenases, and we propose to explore mechanisms of selectivity and proceed with efforts to expand their substrate recognition and biocatalytic properties. Grand Challenge III focuses on natural product discovery and pathway engineering. We have established the technologies and bioinformatics capabilities to readily assemble and mine genomic, and metagenomic datasets from diverse microbiome populations toward natural product gene cluster discovery, which is now poised for heterologous expression in amenable microbial hosts. The next wave of progress will rely on ready identification of the most novel pathways, and our ability to express them using facile synthetic biology methods. We plan to attack these problems with utmost energy and determination to gain access to important compounds with valuable medicinal properties.