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

ABSTRACT: 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 proxi

SUBMITTER: Somvilla I 

PROVIDER: S-EPMC12150331 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature