Biosynthetic studies on the heterocycle-containing thiopeptide natural products

描述(申请人提供)：杂环是一组官能团，通常存在于微生物产生的小分子中，包括抗生素和抗癌剂。在这里，我们打算研究一类含杂环的抗菌化合物--噻唑基多肽的生物合成。了解这类化合物的生物合成起源，不仅将为这些分子的新类似物的组合生物合成提供基础，而且将有助于发现新的含有杂环的天然产物，这些产物可能具有更好的药代动力学性质。过去，研究人员一直没有找到负责产生噻唑基多肽的基因簇。我们最近做了一个令人惊讶的观察，硫霉素是从核糖体合成的短肽中衍生出来的，这些短肽经过高度的翻译后修饰来产生具有生物活性的天然产物。本提案描述的具体目标是：(1)阐明蜡状芽孢杆菌生物合成硫氧西林过程中翻译后修饰的顺序。要阐明硫氧西林生物合成的机制，需要对每个假想的生物合成基因进行系统的双交叉敲除。合成中间体将通过高分辨率质谱仪(HRMS)和核磁共振分析对高效液相纯化的化合物进行鉴定。(2)考察该体系的底物范围，为生物合成新的硫肽类似物奠定基础。编码硫代青霉素肽骨架的结构基因将被丙氨酸扫描，以确定可以被取代的位置，而不会扰乱多肽的翻译后修饰。(3)通过对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有杂环的天然产物的新类别。我们利用通过鉴定硫氧西林基因簇获得的信息，在其他芽孢杆菌菌株中发现了一个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液来鉴定这些新基因簇的产物。我们将使用2D-核磁共振和HRMS来解析新分子的结构。 公共卫生相关性：细菌对抗菌剂的耐药性仍然是对公共健康的主要威胁。噻唑基多肽是一类对多种革兰氏阳性菌有效的抗菌化合物，但具有较差的药代动力学特性。了解噻唑基多肽的生物合成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性质的类似物，从而加强我们的无耐药性抗生素的武器库。

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.