Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule. Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant. A novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.

中文翻译：

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用于高通量细胞培养和酶分析的稳定和选择性渗透水凝胶微胶囊。

过去二十年来，人工微区室内生化反应体积的小型化一直是多种催化剂定向进化的关键驱动力。通常，单细胞与荧光底物共同划分在油包水乳液液滴内，荧光底物的转化可以识别具有改进性能的催化剂。然而，基于乳液滴的技术阻止细胞增殖至高密度，并排除了需要交换小分子底物的生化反应的可行性。在这里，我们报告了一种高通量筛选方法的开发，该方法解决了这些缺点，并依赖于新型选择性渗透聚合物水凝胶微胶囊。采用射流破碎和逐层（LBL）技术成功构建了具有选择渗透性的空心聚电解质涂层壳聚糖海藻酸盐微胶囊（HC-PCAM）。我们表明，HC-PCAM 可作为单细胞封装的微型容器，使细胞生长至高密度，并使细胞裂解生成单克隆细胞裂解液室，适合使用大型颗粒分选机 (COPAS) 进行高通量分析。使用痤疮丙酸杆菌成键酶分选酶 F 催化的转肽反应证明了使用 HC-PCAM 作为交换小分子底物的反应室的可行性。微胶囊周围的聚电解质壳允许荧光标记的肽底物进入微胶囊并参与细胞裂解时保留在胶囊内的细胞内表达的分选酶催化的转肽反应。荧光转肽产物在微胶囊内的特异性保留使得分选酶活性能够与荧光读数联系起来，并允许表达野生型SrtF的微胶囊与表达无活性变体的微胶囊清晰分离。开发了一种基于聚合物水凝胶微胶囊的新型方法，该方法允许基于单细胞封装进行高通量分析。该方法已针对分选酶的转肽活性进行了验证，并且是筛选分选酶、其他成键酶以及定向进化实验中的结合亲和力文库的强大工具。此外，封装微菌落的选择性渗透微胶囊为制备新型笼状生物催化剂和生物传感器提供了一种新的有效手段。