Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate...

Methanol is a promising one-carbon feedstock for biomanufacturing, which can be sustainably produced from carbon dioxide and natural gas. However, the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide (NAD⁺)-dependent methanol dehydrogenase (Mdh) that oxidizes methanol to formaldehyde. Herein, the neutrophilic and mesophilic NAD⁺-dependent Mdh from Bacillus stearothermophilus DSM 2334 (Mdh_Bs) was subjected to directed evolution for enhancing the catalytic activity. The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants. Mdh_Bs variants with up to 6.5-fold higher K_cat/K_M value for methanol were screened from random mutation libraries. The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity. The beneficial T153P mutation changes the interaction network of this residue and breaks the α-helix important for substrate binding into two short α-helices. Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_Bs, and this study provides an efficient strategy for directed evolution of Mdh.