Issue 25, 2022

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) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115–373 Da. An F186L CDPS mutant was isolated to produce cyclo(L-Phe–L-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
22 Mär 2022
Accepted
01 Jun 2022
First published
02 Jun 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2022,13, 7581-7586

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

S. Zhang, J. Zhu, S. Fan, W. Xie, Z. Yang and T. Si, Chem. Sci., 2022, 13, 7581 DOI: 10.1039/D2SC01637K

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