Ultrahigh-Throughput Activity Engineering of Promiscuous Amidases through a Fluorescence-Activated Cell Sorting Assay

Ultrahigh-throughput methods such as flow cytometry are ideal tools for the directed evolution of enzymes by enabling the screening of up to 10⁹ 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 S-transferase. To demonstrate the applicability of the FACS-based assay, we engineered an amidase from Sphingomonas alpina (SaAmd) by screening combinatorial libraries with multiple amino acid positions randomized simultaneously. SaAmd 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 N-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.