Profilingof Microbial Colonies for High-ThroughputEngineering of Multistep Enzymatic Reactions via Optically GuidedMatrix-Assisted Laser Desorption/Ionization Mass Spectrometry

摘要

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolutionMS and tandem MS were readily performed on the same sample target.Separately, relative ion intensities of rhamnolipid congeners withvarious lipid moieties were evaluated to engineer enzymatic specificity.The glycolipid profiles of each colony were overlaid with opticalimages to facilitate the recovery of desirable mutants. For both theantibiotic and rhamnolipid cases, large populations of colonies wererapidly surveyed at the molecular level, providing information-richinsights not easily obtained with traditional screening assays. Utilizingstandard microbiological techniques with routine microscopy and MALDI-ToFMS instruments, this simple yet effective workflow is applicable fora wide range of screening campaigns targeting multistep enzymaticreactions.