Somvilla Ina, Meinert Hannes, Cziegler Clemens, Gökler Tobias, Berner Christoph F, Wolfgramm Hannes, Branson Yannick, Conibear Anne C, Völker Uwe, Badenhorst Christoffel P S, Bayer Thomas, Bornscheuer Uwe T
Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Greifswald 17487, Germany.
Institute of Applied Synthetic Chemistry, TU Wien, Vienna 1060, Austria.
ACS Catal. 2025 May 12;15(11):8902-8912. doi: 10.1021/acscatal.5c01903. eCollection 2025 Jun 6.
Ultrahigh-throughput methods such as flow cytometry are ideal tools for the directed evolution of enzymes by enabling the screening of up to 10 variants per day. In this study, we developed an assay based on fluorescence-activated cell sorting (FACS) for the detection and engineering of amidase activity in whole cells. The assay establishes a stable genotype-phenotype linkage by coupling coumarin-based hydrolysis products to intracellular glutathione via a recombinantly coexpressed glutathione -transferase. To demonstrate the applicability of the FACS-based assay, we engineered an amidase from (Amd) by screening combinatorial libraries with multiple amino acid positions randomized simultaneously. Amd variants containing proximal double mutations exhibited not only almost 5-fold improved activity against structurally different amide substrates but also coevolved promiscuous carbamate- and ester-hydrolyzing activities, which exceeded the wildtype activity up to 6-fold. Importantly, triple variants featuring distal mutations in three highly flexible loop regions, displayed up to 16-fold enhanced specific activities toward small molecules containing highly stable -aryl amide and carbamate bonds. These motifs are commonly used as protecting groups for amines in organic synthesis but can also be found in environmental contaminants like pesticides and plastic waste. Therefore, the developed FACS-assisted assay has great potential to accelerate the engineering of amidases for versatile biotechnological applications.
诸如流式细胞术这样的超高通量方法是通过每天能够筛选多达10种变体来进行酶的定向进化的理想工具。在本研究中，我们开发了一种基于荧光激活细胞分选（FACS）的测定法，用于检测和改造全细胞中的酰胺酶活性。该测定法通过将基于香豆素的水解产物经由重组共表达的谷胱甘肽 - 转移酶与细胞内谷胱甘肽偶联，建立了稳定的基因型 - 表型联系。为了证明基于FACS的测定法的适用性，我们通过同时随机化多个氨基酸位置来筛选组合文库，对一种酰胺酶（Amd）进行了改造。含有近端双突变的Amd变体不仅对结构不同的酰胺底物表现出近5倍的活性提高，而且还共同进化出混杂的氨基甲酸酯和酯水解活性，其超过野生型活性高达6倍。重要的是，在三个高度灵活的环区域具有远端突变的三重变体，对含有高度稳定的芳基酰胺和氨基甲酸酯键的小分子表现出高达16倍的比活性增强。这些基序通常在有机合成中用作胺的保护基团，但也可以在农药和塑料废物等环境污染物中找到。因此，所开发的FACS辅助测定法具有巨大潜力，可加速酰胺酶的工程改造以用于多种生物技术应用。
