Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening. Issue 25 (14th June 2022)

• Record Type: Journal Article
• Authors: Zhang, Songya; Zhu, Jing; Fan, Shuai; Xie, Wenhao; Yang, Zhaoyong; Si, Tong
• Is Part Of: Chemical science. Volume 13:Issue 25(2022)
• Page Start: 7581
• Page End: 7586
• Publication Date: 2022-06-14
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2sc01637k
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : A robotic workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS 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.