Biosynthetic studies on the heterocycle-containing thiopeptide natural products

描述（由申请人提供）：杂环是一组常见于微生物产生的小分子（包括抗生素和抗癌剂）中的官能团。在此，我们建议研究噻唑基肽，一类含杂环的抗菌化合物的生物合成。了解这个家族的化合物的生物合成的起源，不仅提供了这些分子的新的类似物的组合生物合成的基础，但将有助于发现新的类杂环的天然产物，可能会表现出改善的药代动力学特性。过去，研究人员一直未能找到产生噻唑基肽的基因簇。我们最近作出了令人惊讶的观察，thiocillins是来自核糖体合成的短肽，经过高度的翻译后修饰，产生生物活性的天然产物。本研究的具体目的是：（1）阐明蜡状芽孢杆菌中硫代青霉素生物合成过程中的翻译后修饰顺序。阐明硫代西林生物合成的机制将需要系统的双交换敲除每个假设的生物合成基因。将通过HPLC纯化化合物的高分辨率质谱（HRMS）和NMR分析，在敲除菌株中鉴定合成中间体。(2)研究该系统的底物范围，以生物合成产生新的硫肽类似物。将对编码硫代西林的肽骨架的结构基因进行丙氨酸扫描，以确定可以被取代而不破坏肽的翻译后修饰的位置。(3)通过微生物基因组的生物信息学分析，发现这一天然产物家族的新成员以及含杂环天然产物的新类别。我们使用通过鉴定硫代青霉素基因簇获得的信息来发现其他芽孢杆菌菌株中相关化合物的基因簇。我们将通过系统比较含有相同基因簇的细菌菌株的提取物来鉴定这些新基因簇的产物。我们将使用2D-NMR和HRMS解析新分子的结构。 公共卫生相关性：细菌对抗菌药物的耐药性仍然是对公共卫生的主要威胁。噻唑基肽是一类抗微生物化合物，其对许多革兰氏阳性细菌有效，但表现出差的药代动力学性质。对噻唑基肽生物合成的理解将使我们能够识别新的生物活性分子，并产生具有改进的药代动力学特性的类似物，从而加强我们的无耐药性抗生素库。

DESCRIPTION (provided by applicant): Heterocycles are a set of functional groups commonly found in microbially-produced small molecules, including antibiotics and anticancer agents. Herein, we propose to study the biosynthesis of the thiazolylpeptides, a class of heterocycle-containing antimicrobial compounds. An understanding of the biosynthetic origins of this family of compounds will not only provide the foundation for combinatorial biosynthesis of novel analogues of these molecules, but will aid in the discovery of new classes of heterocycle-containing natural products which may exhibit improved pharmacokinetic properties. The gene clusters responsible for producing the thiazolyl peptides have eluded researchers in the past. We recently made the surprising observation that the thiocillins are derived from ribosomally synthesized short peptides that undergo a high degree of posttranslational modification to produce the biologically active natural products. The specific aims described in this proposal are: (1) To elucidate the order of post-translational modifications during thiocillin biosynthesis in Bacillus cereus. Elucidation of the mechanism of thiocillin biosynthesis will require systematic double crossover knockouts of each hypothetical biosynthetic gene. Synthetic intermediates will be identified in knockout strains through high resolution mass spectrometry (HRMS) and NMR analysis of compounds purified by HPLC. (2) To examine the substrate scope of this system in order to biosynthetically generate new thiopeptide analogs. The structural gene which encodes the peptide backbone of thiocillin will be alanine-scanned to determine positions that can be substituted without disrupting posttranslational modification of the peptide. (3) To discover new members of this family of natural products as well as novel classes of heterocycle-containing natural products through bioinformatic analysis of microbial genomes. We used the information garnered through identification of the thiocillin gene cluster to discover a gene clusters of related compounds in other Bacillus strains. We will identify the products of these novel gene clusters through systematic comparison of extracts from bacterial strains which contain the same gene cluster. We will solve the structure of the new molecule using 2D-NMR and HRMS. PUBLIC HEALTH RELEVANCE: Bacterial resistance to antimicrobial agents continues to be a major threat to public health. The thiazolyl peptides are a class of antimicrobial compounds which are effective against a number of Gram-positive bacteria, but exhibit poor pharmacokinetic properties. An understanding of thiazolyl peptide biosynthesis will enable us to identify new biologically active molecules, and generate analogues with improved pharmacokinetic properties, and thus strengthen our arsenal of resistance-free antibiotics.