Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (application 1) and to perform directed laccase evolution through a semipurification process in 96-well microtiter plate (MTP) (application 2). A semipurification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI, which acts as adhesion promoter. To validate the SortEvolve screening platform for both applications, three site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) from Staphylococcus aureus (application 1) and two SSM libraries of the copper efflux oxidase (CueO laccase) from Escherichia coli (application 2) were generated at literature reported positions. After screening and rescreening, an array of Sa-SrtA variants (including the previously reported P94S, D160N, and D165A) and CueO variants (including the previously reported D439A and P444A) were identified. Further recombinant Sa-SrtA variant P94T/D160L/D165Q and CueO variant D439V/P444V were characterized with 22-fold and 103-fold improvements in catalytic efficiency compared with corresponding wild-types, respectively. An important advantage of the SortEvolve screening platform in comparison to many MTP-based screening systems is that the background noise was minimized (decreased 20-fold; application 2) due to the employed semipurification process. In essence, SortEvolve provides a universal surface-functionalized screening platform for sortases and enzymes in which especially background activity can be minimized to enable successful directed evolution campaigns.

中文翻译：

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分选酶介导的高通量筛选平台，用于定向酶进化

在材料科学和生物催化中，分选酶催化的连接已成为肽和蛋白质位点特异性连接的强大工具。在这项工作中，已开发出定向分选酶进化策略（SortEvolve）作为通用的高通量筛选（HTS）平台，以提高分选酶A的活性（应用1）并通过半纯化过程在96孔中进行定向的漆酶进化。微量滴定板（MTP）（应用2）。聚丙烯MTP（PP-MTP）的半纯化过程是通过锚定肽LCI（起粘合促进剂作用）实现的。为了验证这两种应用的SortEvolve筛选平台，使用了来自金黄色葡萄球菌的分选酶A（Sa-SrtA）的三个位点饱和诱变（SSM）文库（应用程序1）和两个来自大肠杆菌的铜外排氧化酶（CueO漆酶）的SSM文库（应用程序2）是在文献报道的位置生成的。筛选和重新筛选后，鉴定了一系列Sa-SrtA变体（包括先前报道的P94S，D160N和D165A）和CueO变体（包括先前报道的D439A和P444A）。与相应的野生型相比，进一步的重组Sa-SrtA变体P94T / D160L / D165Q和CueO变体D439V / P444V的催化效率分别提高了22倍和103倍。与许多基于MTP的筛查系统相比，SortEvolve筛查平台的一个重要优势在于，由于采用了半纯化工艺，背景噪音得以最小化（降低了20倍；应用2）。在本质上，