中文摘要：噬菌体编码的细胞裂解酶是一类降解肽聚糖的新型抗菌酶。细胞裂解酶的标志性特征是模块化组成，通过基于细胞裂解酶和非细胞裂解酶模块的酶候选蛋白的工程设计，可以优化抗菌和药理特性。在这方面，最近引入的多功能技术允许以预制构建块存储库为基础快速构建大型模块化细胞裂解酶文库。本研究以肺炎克雷伯菌为目标，高通量构建并筛选了5个不同构型的组合细胞裂解酶文库。详细分析940个变体的活性分布和74个抑制肺炎克雷伯菌生长的热门基因的测序数据可能与特定的设计规则有关。特异性外膜通透肽（OMPs）和酶活性域（EADs）在热门基因中明显过高，而细胞壁结合域（CBDs）的比例相同。特别是带有构型（OMP链接器CBD EAD）和反向构型（CBD EAD链接器OMP）文库产生了最活跃的变体，在其余变体之上出现了可识别的变体簇。本文采用的方法为从不同构型和组成的文库开始发现工程化细胞裂解酶的活动提供了蓝图。
外文摘要：Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP-linker-CBD-EAD) and the inverse configuration (CBD-EAD-linker-OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.
外文关键词：lysin；bacteriophage；VersaTile；Klebsiella pneumoniae；protein engineering
作者：Duyvejonck, L；Gerstmans, H；Stock, M；Grimon, D；Lavigne, R；Briers, Y
作者单位：Univ Ghent；Katholieke Univ Leuven
期刊名称：ANTIBIOTICS-BASEL
期刊影响因子：3.893
出版年份：2021
出版刊次：3
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