Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening.

ABSTRACT: Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM)

SUBMITTER: Zhang S 

PROVIDER: S-EPMC9241961 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature