(569c) A Structure-Based High-Throughput Screening Method for Multi-Step Enzymatic Reactions Using Optically-Guided Mass Spectrometry Profiling of Microbial Colonies

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has been used for rapid phenotyping of enzymatic activities but mainly limited to single-step conversions. To engineer a multi-step reaction, however, modified intermediates must be accepted at each step of the catalytic sequence to obtain a final product. Engineering an individual enzyme in isolation ignores possible downstream effects, and MSI screening platforms primarily designed for single-step enzymatic reactions may be ill-suited for engineering multi-step pathways.

Herein we report a label-free, structure-based method for high-throughput engineering of multi-step biochemical reactions, based on optically-guided MALDI-MS analysis of bacterial colonies. The use of bacterial cells provides containment of multiple enzymes and access to substrates and cofactors via metabolism. Automatic MALDI-MS acquisition at a rate of 1~2.5 s per colony using machine vision for randomly distributed colonies allows simple procedures to prepare strain libraries without advanced liquid handling. To process the resulting large mass spectral datasets, computational tools were developed to survey structural variations and/or quantitative changes of analogs or congeners. Screening results are visualized in a manner similar to classical, colorimetric assays, allowing straightforward interpretation for researchers with limited MS experience.

The MALDI-MS profiling approach was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. For analogs of a peptidic antibiotic, plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding successfully enumerated structural diversity and clustered colonies with isobaric residue substitutions. Follow-up analysis, including high-resolution MS and tandem MS, was readily performed from the same sample target following MALDI-MS screening. We identified both known and new plantazolicin analogs, and all assignment based on MS results were consistent with subsequent DNA sequencing analyses of the precursor peptide library. Separately, in the context of rhamnolipid synthesis, relative intensities of congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate mutant recovery. Enzyme variants with altered specificities were successfully isolated to produce rhamnolipid mixtures with custom congeners compositions. In both cases, large populations of colonies were rapidly characterized at the molecular level, providing information-rich insights not easily accessible with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-MS instruments, this simple yet effective workflow is applicable for a wide range of structure-based screening applications in microbial systems.