DataSheet1_Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant.PDF

Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS (MaPylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of MaPylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve MaPylRS to efficiently incorporate Nε-Boc-l-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRSopt) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRSopt can be used to enhance previously engineered PylRS constructs such as MaPylRSN166S, and PylRSopt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.吡咯赖氨酰-tRNA合成酶（Pyrrolysyl-tRNA synthetase，PylRS）常被用于将非经典氨基酸（noncanonical amino acids，ncAAs）定点掺入蛋白质中。近期，研究者对甲烷嗜甲基杆菌PylRS（Methanomethylophilus alvus PylRS，MaPylRS）的活性位点开展理性工程改造，拓展了其底物适配范围，使其可高效掺入以往难以引入的非经典氨基酸。然而，目前尚未见报道称活性位点以外的突变可增强MaPylRS的酶学性能。本研究借助噬菌体辅助非连续进化（phage-assisted non-continuous evolution，PANCE）技术对MaPylRS进行定向进化，以实现Nε-叔丁氧羰基-L-赖氨酸（Nε-Boc-L-lysine，BocK）的高效掺入。定向进化筛选得到多个位于活性位点之外的突变位点，这些突变可显著提升该酶的催化活性。研究人员将最优的有效突变组合，构建出新型PylRS变体PylRSopt，该变体对多种赖氨酸及苯丙氨酸衍生物均具备优异的催化活性与选择性。PylRSopt所携带的突变可用于增强已有的工程化PylRS构建体（如MaPylRSN166S），且该变体可适配双非经典氨基酸掺入的应用场景，大幅提升此类靶蛋白的表达产量。