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High-Throughput, Lysis-Free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2019-12-04 , DOI: 10.1021/acssynbio.9b00274 Bert van Loo 1 , Magdalena Heberlein 1, 2 , Philip Mair 2 , Anastasia Zinchenko 2 , Jan Schüürmann 3 , Bernard D G Eenink 1 , Josephin M Holstein 2 , Carina Dilkaute 3 , Joachim Jose 3 , Florian Hollfelder 2 , Erich Bornberg-Bauer 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2019-12-04 , DOI: 10.1021/acssynbio.9b00274 Bert van Loo 1 , Magdalena Heberlein 1, 2 , Philip Mair 2 , Anastasia Zinchenko 2 , Jan Schüürmann 3 , Bernard D G Eenink 1 , Josephin M Holstein 2 , Carina Dilkaute 3 , Joachim Jose 3 , Florian Hollfelder 2 , Erich Bornberg-Bauer 1
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Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of an ultra high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure, we quantitatively screened >105 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Compartmentalization of the fluorescent reaction product with living E. coli cells autodisplaying the sulfatase variants ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1 variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between α-helices in the C-terminal oligomerization region, suggesting a previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.
中文翻译：
使用微滴中的大肠杆菌自动展示技术进行高通量，无裂解的硫酸酯酶活性筛选。
朝着改善的催化性能方向定向发展的酶已成为蛋白质工程中的强大工具。为了有效进行定向进化运动，需要使用高通量筛选。在这项研究中，我们描述了通过结合基于微滴的单变量活性分选和大肠杆菌自动展示筛选超高通量无裂解程序来筛选硫酸酯酶活性的方法。对于4步筛选过程中的第一步，我们从大肠杆菌中筛选出了多于105种来自大肠杆菌（Salicibacter pomeroyi）（SpAS1）的同型二聚芳基硫酸酯酶变体，以利用荧光激活的液滴分选方法改善硫酸酯酶的活性。荧光反应产物与活E的区室化 自动展示硫酸酯酶变体的大肠埃希菌细胞确保了液滴分选过程中基因型和表型的连续连接，并通过分选后的细胞简单再生长而直接恢复。在活细胞上使用自动显示简化了筛选过程中所有步骤中液体处理的程度，并将其降低到仅混合底物和细胞的单一事件。由于微滴分选，表观改良变体的百分比富集了> 10倍。我们最终确定了25个SpAS1变异体，它们对4-硝基苯基硫酸盐（最多6.2倍）和/或荧光素二硫酸盐（最多30倍）的性能有所改善。在SpAS1变体中，对于大体积的二氟荧光素，其性能有所改善，
更新日期：2019-12-04
中文翻译：
使用微滴中的大肠杆菌自动展示技术进行高通量，无裂解的硫酸酯酶活性筛选。
朝着改善的催化性能方向定向发展的酶已成为蛋白质工程中的强大工具。为了有效进行定向进化运动，需要使用高通量筛选。在这项研究中，我们描述了通过结合基于微滴的单变量活性分选和大肠杆菌自动展示筛选超高通量无裂解程序来筛选硫酸酯酶活性的方法。对于4步筛选过程中的第一步，我们从大肠杆菌中筛选出了多于105种来自大肠杆菌（Salicibacter pomeroyi）（SpAS1）的同型二聚芳基硫酸酯酶变体，以利用荧光激活的液滴分选方法改善硫酸酯酶的活性。荧光反应产物与活E的区室化 自动展示硫酸酯酶变体的大肠埃希菌细胞确保了液滴分选过程中基因型和表型的连续连接，并通过分选后的细胞简单再生长而直接恢复。在活细胞上使用自动显示简化了筛选过程中所有步骤中液体处理的程度，并将其降低到仅混合底物和细胞的单一事件。由于微滴分选，表观改良变体的百分比富集了> 10倍。我们最终确定了25个SpAS1变异体，它们对4-硝基苯基硫酸盐（最多6.2倍）和/或荧光素二硫酸盐（最多30倍）的性能有所改善。在SpAS1变体中，对于大体积的二氟荧光素，其性能有所改善，
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