Robert A. Shepherd, Manasa Ramachandra, Austin R. Hopiavuori, Marla Jones, Conrad A. Fihn, Alex J. Tabag, Ananya Manjunath, Limar Y. Gad, Chloe R. Whipple, Shaun M. K. McKinnie, Laura M. Sanchez
The growing field of biocatalysis relies on the generation of large, genetically diverse libraries for downstream colorimetric, fluorogenic, or chromatographic screening. However, challenges remain when assessing enzyme variants in a high-throughput manner whose transformations yield isomeric low-molecular-weight products. The kainoid synthase subfamily of Fe/αKG-dependent dioxygenases produces the isomeric neurochemicals kainic acid (KA) and KA lactone (KAL) in a range of yields and ratios. To enable an improved throughput screening of engineered kainoid synthases, we developed a matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry (MALDI-TIMS-MS) platform to directly detect regioisomeric products with near-baseline resolution from bacterial colonies. Screening of 1,054 genetically diversified KabC variants identified seven with improved KAL formation, while retaining favorable expression profiles and improved in vitro stabilities. This workflow establishes MALDI-TIMS-MS as a promising platform for high-throughput isomeric product screening and provides unique enzyme constructs to probe structure-function relationships and for further biocatalytic development.