Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

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-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

“…1a) [12]. MALDI MS is well suited for rapid inspection of a large number of biological samples because of its simple sample preparation, high salt tolerance, and a wide coverage of diverse biomolecules [12,25,26]. Traditionally, macromolecules such as proteins, lipids, and glycans are the main targets for MALDI MS, but small metabolites are increasingly analyzed as well (Table 1) [32].…”

“…When coupled with machine vision, laser sampling can be programmed to target randomly located objects such as microbial colonies [33]. This development enabled rapid engineering of multi-step enzymatic pathways using microbial cells as reaction vessels [26].…”

“…To overcome such limitations, MALDI MS imaging can provide unparalleled speed and selectivity to distinguish lipid congeners of various chain lengths based on mass differences. For example, we recently developed optically guided MALDI MS to profile the chemical content of microbial colonies at a rate of~5 s [26]. The total and relative abundance of various rhamnolipid congeners were rapidly quantified by monitoring the ion intensities at corresponding m/z values (Fig.…”

“…Using this method, we were able to rapidly screen thousands of mutant strains for directed enzyme evolution [26]. However, biofuel-relevant lipid molecules, including free fatty acids, fatty alcohols, and alkenes, are challenging targets for MALDI MS.…”

“…Alternatively, ESI MS assays may also be developed to screen fatty-acid derived products. For example, lipid [26]. A Chemical structure of the main rhamnolipid molecules produced from recombinant microbial cells.…”