Identification of overoxidizing and non-overoxidizing NAD-dependent methanol dehydrogenases and implications for synthetic methylotrophy

Synthetic methylotrophy offers opportunities for sustainable chemical and biofuel production. While recently established methylotrophic E. coli can grow on methanol, undesirable formate accumulation occurs during growth and bioproduction. Here, we show that NAD-dependent methanol dehydrogenase Mdh2 from Cupriavidus necator inherently overoxidizes methanol to formate, a trait we find to be widespread among NAD-dependent Mdh enzymes. In contrast, Mdh/Mdh1 enzymes from Bacillus methanolicus exclusively oxidize methanol to formaldehyde without overoxidation, as we validate in vitro for Mdh Bm MGA3 with and without activator protein Act. Since only formaldehyde is assimilated via the ribulose monophosphate pathway, this explains the physiological role of Mdh/Mdh1 paralogs in natural methylotrophs and highlights the importance of selecting appropriate Mdh variants for synthetic methylotrophy. We demonstrate methanol-dependent growth using non-overoxidizing Mdh Bm MGA3, strongly reducing formate accumulation and carbon loss. Our findings reveal a characteristic of NAD-dependent Mdh enzymes and provide insights for engineering synthetic methylotrophs.