达托霉素C结构域dptC1的底物选择性分子机制解析

Non-ribosomal Peptides Synthetases (NRPS) is a multimodular megaenzyme that can catalyze nonribosomal peptides with diverse bioactivities. The daptomycin is biosynthesized by NRPS in Streptomyces roseosporus, forming the N-decanoyl peptide antibiotic serving as an important clinical antimicrobial drug. The C domain (dptC1) is proposed to condensate different N-fatty acyl chain as a result of multicomponent mixtures. In structure-activity relationship, it was verified the N-decanoyl modification is crucial for antimicrobial activity. Hence the substrate selectivity of dptC1 plays a key role in target N-decanoyl biosynthesis. As far as reported, the substrate selectivity of C domain is remaining under explored. In this study, we will construct the in vitro system of dptC1, and investigate the dynamics behavior of dptC1 with various fattyl acyl substrates. Then combining with protein engineering and computer simulation strategies, the rational analysis was carried on to locate the key residues and area for substrate binding. These candidate residues will be applied with saturation mutations to reveal the molecular mechanism of substrate selectivity, and to obtain an artificial element highly catalyzing decanoyl substrate. Finally, the daptomycin overproduction could be achieved through test and regulate the artificial element in vivo, by means of overcoming the non-specific condensation of decanoyl substrate. Our study will uncover the molecular mechanism of C domain guiding rational design, as well as provide theoretical support for other NRPS pathways engineering and optimization in further.

非核糖体肽合成酶（NRPS）是由多结构域组成的酶复合体，可催化合成活性丰富的多肽类次级代谢产物。达托霉素由玫瑰孢链霉菌中NRPS合成的N-癸酰环肽类抗生素，是临床上重要的抑菌药物。其C结构域（dptC1）催化加载不同N-脂酰底物，产生多组分混合物，且构效关系发现N-癸酰修饰对活性至关重要，说明dptC1的脂酰底物选择性对目标N-癸酰合成效率具有重要影响。目前C结构域的底物选择性分子机制未见报道。本项目拟建立dptC1体外催化体系，以不同脂酰底物进行动力学研究，并利用蛋白质工程和计算机模拟等技术进行理性分析，定位酶底物结合的关键残基和区域，通过饱和突变揭示底物选择分子机制，并获得高效加载癸酰底物的人工元件，最后调控并提高人工元件的体内适配性，打破体内癸酰底物加载的“非专一性”，实现达托霉素高产优产。本项目将揭示C结构域分子作用机制并指导理性设计，为其他NRPS途径的改造及优化奠定理论。

利用酶学、蛋白质工程等研究方法，以达托霉素生物合成中dptC1为研究对象，揭示NRPS中C结构域的底物选择性机制，研究dptC1的体外动力学，尤其是对癸酰底物的催化能力，并在此基础上分析酶底物选择的关键残基和区域，进行目的性改造，定向、有效的提高酶对癸酰底物的底物选择性及催化效率，最终在体内开发原位替换的基因组编辑方法，测试人工元件的体内适配性，实现达托霉素的高产优产。