利用随机化的结构域重排构建高活性、低耐药和广谱的多模块细菌裂解酶

With the wide spread application of antibiotics, drug resistant pathogens are becoming more and more popular. What make the situations worse are the difficulties facing the development of novel antibiotics. Because of different targets and mechanism, antibacterial lytic enzymes are being developed as supplements or even alternatives of conventional antibiotics. However, the natural antibacterial lytic enzymes have defects in their activity, resistance and antibacterial spectrum. Based on the modular structure and the latest understanding of the relationship between structure and activity gained from the PlyC lytic enzyme, we proposed a multi-modular re-engineering strategy. Using isocaudarners mediated directed ligation, we were able to accomplish domain shuffling in a completely randomized manner, and thus mimicked the natural evolution process. Novel enzymes with improved activity, reduced resistance and broadened antibacterial spectrum can be identified from the multi-modular antibacterial lytic enzyme library by high throughput screening. These enzymes have promising applications in infection treatment, environmental disinfection and food preservation. Moreover, the technology established in this study can be generally applicable to combat the emerging pathogens.

随着抗生素的广泛应用，耐药致病菌正越来越普遍。与此相反，新型抗生素的研制却举步维艰。由于与抗生素具有不同的作用靶点和机制，细菌裂解酶有望成为传统抗生素的补充甚至替代。然而，天然细菌裂解酶在活性，耐药性以及抗菌谱上存在缺陷。基于细菌裂解酶模块化的结构特点和对于PlyC裂解酶结构和功能的最新认识，我们提出了多模块化的改造策略。利用同尾酶介导的定向连接技术，实现了多个结构域的完全随机的重排，再现了细菌裂解酶的自然进化过程。通过对多模块细菌裂解酶文库的高通量筛选，可以获得在活性，耐药性以及抗菌谱上显著优于天然细菌裂解酶的人工酶。上述酶在相关致病菌引起的感染治疗，环境消洗以及食品防腐等领域具有良好的应用前景。而且，本研究建立的关键技术还具有通用性和平台性的特点，可以用于新发致病菌裂解酶的快速进化。

细菌裂解酶有望成为一种新型的治疗传统抗生素耐药菌的药物。以lysostaphin金黄色葡萄球菌最为研究对象，我们在以下三个方面进行了系统的研究：（1）通过基因序列和蛋白序列的优化，实现了均一的非糖基化的lysostaphin在巴斯德毕赤酵母中的分泌表达；（2）利用十余种具有代表性的细胞壁结合模块对lysostaphin进行了系统的优化，提示了细菌裂解酶的催化结构域和结合结构域的共进化机制；（3）利用血清白蛋白融合技术对lysostaphin的体内药代动力学进行了优化，发现具有平衡的药效/药代动力学性质的融合蛋白具有最高的体内疗效。