Collaborative Research: RUI: Elucidating Design Rules for non-NRPS Incorporation of Amino Acids on Polyketide Scaffolds

越来越多的细菌对目前的抗生素产生了抗药性。具有抗生素特性的天然化合物在细胞内以模块化的方式组装。模块化的特性允许设计混合途径来产生非天然分子。抗生素分子中单个键形成反应的机制将被研究。其目的是发展一定程度的洞察力，从而能够重组构建块以生产新型抗生素。这个概念类似于分子构建模块。此外，这项工作将为培养本科生进行生物技术研究和向他们的社区传播科学奠定基础。该项目将试图揭示促进独特天然产物生产的新型酶机制。具体来说，这项工作将阐明氨基酸在聚酮支架上结合的机制基础。该项目重点研究模型分子TLN-05220，它是一种棘孢霉素型抗生素，对几种耐甲氧西林金黄色葡萄球菌和耐万古霉素肠球菌（VRE）具有抗菌活性，并对几种人类肿瘤细胞系具有细胞毒活性。TLN-05220生物合成中的单个基因功能将通过基因敲除在野生型生产生物体中进行鉴定。潜在的底物结合位点和氨基酸结合酶的催化残基将使用同源性建模来确定。单个酶的表达和表征将阐明氨基酸融入天然产物结构的机制。产生TLN-05220的最低必要基因将被克隆并在异源宿主中表达。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

More bacteria have become resistant to current antibiotics. Natural compounds with antibiotic properties are assembled in a modular fashion inside cells. The modular nature allows for the design of hybrid pathways to produce non-natural molecules. The mechanisms of individual bond-forming reactions in an antibiotic molecule will be investigated. The intention is to develop a degree of insight that would enable the shuffling of building blocks to produce novel antibiotics. The concept is akin to molecular building blocks. In addition, this work will provide a foundation for training undergraduate students in conducting biotechnological research and communicating science to their community. The project will attempt to uncover novel enzymatic mechanisms that facilitate production of unique natural products. Specifically, this work will elucidate mechanistic underpinnings of amino acid incorporation on polyketide scaffolds. The project focuses on the model molecule TLN-05220, which is an Echinosporamicin-type antibiotic with antibacterial activity against several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. Individual gene functions in TLN-05220 biosynthesis will be identified using gene knockouts in the wild-type producing organism. Potential substrate binding sites and catalytic residues of amino acid-incorporating enzymes will be identified using homology modeling. Expression and characterization of individual enzymes will elucidate mechanism(s) of amino acid incorporation into natural product structures. The minimally necessary genes to produce TLN-05220 will be cloned and expressed in a heterologous host.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.