Development of improved methanol dehydrogenases using directed evolution and biological methanol sensor system for the elimination of formaldehyde

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Jiyeun Yi, Dae-Hee Lee, Ha Seong Kim, Seung Goo Lee, Bong Hyun Sung

South Korea

Formaldehyde is an important organic precursor to many materials and chemical compounds. Despite its widespread use, exposure to formaldehyde is a significant consideration for human health due to its toxicity and volatility. Methanol dehydrogenase (MDH) is an NAD+-dependent oxidoreductase that catalyzes formaldehyde to methanol reversibly. Reduction activity of MDH is noticeable in two aspects of the elimination of toxic formaldehyde and the production of methanol as an energy source. Herein, we screened improved mutants of Bacillus methanolicus MDH in Esehcichia coli that reduce formaldehyde to methanol effectively with directed evolution and Phichia-based biological methanol sensor system. To examine the resistance to formaldehyde, E. coli strains expressing each mutant were cultured in 3mM formaldehyde, toxic concentration for E. coli. In the best mutant, three phenylalanine residues were substituted to leucine, valine and serine respectively and all the three substitutions were required to increase reduction activity for formaldehyde. Purified mutant converted formaldehyde to methanol approximately 5 times more than wild type. Based on these results, mutant MDH carry out the elimination of formaldehyde efficiently, therefore it could help solve environmental problem such as sick house syndrome.