Multi-objective Engineering of Trimethylamine Monooxygenase for Improved Thermostability and Cofactor Use

Trimethylamine (TMA) is a major contributor to undesirable odours in protein hydrolysates derived from marine by-products, limiting their industrial use. Flavin-containing monooxygenases (FMOs) catalyse the conversion of TMA to the odourless trimethylamine N-oxide (TMAO); however, industrial applications demand enzymes that are both thermally stable and compatible with cost-effective cofactors. A thermostable variant of the Methylophaga aminisulfidivorans FMO (mFMO_20) can function at elevated temperatures but depends exclusively on the expensive and unstable cofactor NADPH. In this study, we investigated whether it is possible to simultaneously enhance thermostability and NADH compatibility using a multi-objective engineering strategy. We first targeted residues in the cofactor binding site of mFMO_20 to restore NADH activity, which had been completely lost despite the wild type enzyme being naturally active with both cofactors. Variants derived from the thermostable scaffold partially recovered NADH activity but showed reduced NADPH activity. Given the wild types inherent NADH compatibility, we next pursued a stability-improvement approach, introducing highly conserved stabilizing mutations. This preserved cofactor competence but produced only modest improvements in thermostability. Finally, by combining physical, evolutionary, and statistical metrics, we obtained variants that retained higher NADPH activity after heat treatment than any previously reported thermostable mutants, while a subset also retained measurable NADH activity before heat treatment. These findings show that combining complementary scoring strategies helps navigate the trade-off between stability and activity; while, robust NADH function under thermal stress remains elusive, with only one variant retaining detectable NADH activity after heat treatment, the results provide valuable insight into the underlying constraints linking stability and cofactor usage and highlights possible directions for engineering FMOs with both enhanced thermostability and cofactor compatibility. Author summaryIn this work, we aimed to improve an enzyme that could be useful in industrial applications but is limited by two common constraints: poor stability at high temperatures and dependence on an expensive cofactor. To make the enzyme more suitable for large-scale applications, we sought to engineer variants that are both more thermostable and compatible with a cheaper cofactor, NADH. For enzyme engineering, we used a strategy that balances several properties rather than prioritizing a single trait. We combined tools that capture evolutionary patterns, protein physics, and AI-based predictions to explore which mutations might provide the right combination of stability and function. Through this approach, we obtained variants with improved heat resistance and higher cofactor activity retention.