Mass Spectrometric Studies of Thiotemplate Biosynthesis

 描述(申请人提供)：随着微生物基因组测序变得越来越普遍，微生物生产天然产品的能力正在得到更好的认识。这一竞争性更新使用最新的计算和表达筛选技术来调查这一能力，并针对从微生物世界发现天然产品的全新平台出现的特定亚类天然产品。这里描述的是对一组特定的天然产物及其基因簇的追求，这些天然产物及其基因簇是从一种新的方法中出现的，该方法实现了生物合成基因簇与其小分子的大规模配对以供发现。从178株放线杆菌的文库中，使用了基因组使能的代谢组学方法(也称为“代谢基因组学”)来正确鉴定天然产物，并将它们与其生物合成基因簇相关联。拟议的研究将这一方法扩展到检测未知的代谢物，坦溴霉素(AIM 1)和利莫沙胺(AIM 2)，每一种都有各自的簇，显示出不寻常的生物合成机制和单体，如新的氨基酸“TAMBROLINE”(见目标1)。在前两个目标中，将研究和定义这些新的天然产物的精确结构、生物合成细节以及在坦溴霉素的情况下-蛋白质结合靶标(S)。在目标3a中，将在电子计算机中挖掘一组新的基因簇-化合物对，用于那些终止于还原酶域的基因簇-化合物对，因此可能包含从硫代模板装配线上还原卸载而来的亲电弹头。目的3b提出了一种扩展这种以基因簇为重点的方法，通过有针对性地挖掘代谢基因组学数据来发现新的生物活性代谢物，以寻找其他已知具有生物活性的药物载体。一种新的多烯抗生素的具体情况是基于序列数据提出的，该序列数据表明在生物合成簇内存在多个脱氢酶结构域。用“代谢基因组学”挖掘微生物世界代表着这项R01资助中过去的活动的重大转变(在过去的授权期内，这项活动导致了18份出版物)。随着一系列新化合物及其生物合成基因簇的掌握，研究人员将以目标模式追踪其中的几个。除了这项提议的直接影响外，这项工作还为天然产品发现中由“组学”驱动的复苏提供了一条重要的前进道路。这种复兴为基于结构的天然产物的发现提供了一条更具确定性的途径，并以该领域以前从未见过的速度提供这些结构(又名。最近的文献中所描述的“高通量发现”)。本质上，拟议的研究将证明将微生物菌株集合规律化到真正的化合物库中的价值，这些化合物已知是表达的，并具有特定的亚结构(例如，亲电弹头)。这些价值主张作为激励理由，将包含独特分子结构的新型发现管道与生物活性检测相结合。

 DESCRIPTION (provided by applicant): As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce natural products is coming into better view. This competing renewal surveys this capacity using the latest in computational and expression screening and targets specific sub-classes of natural products that emerge from a fundamentally new platform for discovery of natural products from the microbial world. Described here is the pursuit of specific set of natural products and their gene clusters which have emerged from a new approach that achieves large-scale pairing of biosynthetic gene clusters with their small molecules for discovery. From a library of 178 actinobacteria strains, a genome-enabled metabolomics approach was used (a.k.a., "metabologenomics") to correctly identify natural products and correlate them with their biosynthetic gene clusters. The proposed research extends this method to examine unknown metabolites, tambromycin (Aim 1) and rimosamides (Aim 2), each with their respective clusters exhibiting unusual biosynthetic mechanisms and monomers, such as the new amino acid, "tambroline" (see Aim 1). In the first two aims, the precise structures, biosynthetic details and in the case of tambromycin-the protein binding target(s) of these new natural products will be studied and defined. In Aim 3a, a set of new gene cluster-compound pairs will be mined in silico for those that terminate in reductase domains, and therefore likely harbor electrophilic warheads from reductive off-loading from thiotemplate assembly lines. Aim 3b proposes an expansion of this gene-cluster focused approach to discovering new bioactive metabolites by targeted mining of metabologenomics data for other pharmacophores known to elicit bioactivity. The specific case of a new polyene antibiotic is put forth based on sequence data indicating the presence of multiple dehydrogenase domains within the biosynthetic cluster. Mining the microbial world with "metabologenomics" represents a major shift from past activities in this R01 grant (which led to 18 publications over the past granting period). With a set of new compounds and their biosynthetic gene clusters in hand, the investigators will pursue several of these in targeted mode. Beyond the direct impact of this proposal, the work provides a major path forward for an 'omics'-driven resurgence in natural products discovery. Such a resurgence promises a more deterministic path for structure- based discovery of natural products and to provide these structures at a rate not seen before in the field (a.k.a. "high throughput discovery" as described in recent literature). In essence, the proposed research will demonstrate the value of regularizing a microbial strain collection into a bona fide library of compounds, which are known to be expressed and possess particular sub-structures (e.g., an electrophilic warhead). These value propositions serve as motivating rationales to merge a new kind of discovery pipeline containing unique molecular structures with assays for bioactivity.