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Sci Rep. 2018 Aug 20;8(1):12483. doi: 10.1038/s41598-018-31001-8.

Development of Bacillus methanolicus methanol dehydrogenase with improved formaldehyde reduction activity.

Yi J1,2, Lee J3,4, Sung BH2,4, Kang DK2,4, Lim G3,4, Bae JH2, Lee SG4,5, Kim SC6, Sohn JH7,8.

Author information

1
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, South Korea.
2
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, South Korea.
3
Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, South Korea.
4
School of Biotechnology, Korea University of Science and Technology, Daejeon, 34113, South Korea.
5
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, South Korea.
6
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, South Korea. sunkim@kaist.ac.kr.
7
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, South Korea. sohn4090@kribb.re.kr.
8
School of Biotechnology, Korea University of Science and Technology, Daejeon, 34113, South Korea. sohn4090@kribb.re.kr.

Abstract

Methanol dehydrogenase (MDH), an NAD+-dependent oxidoreductase, reversibly converts formaldehyde to methanol. This activity is a key step for both toxic formaldehyde elimination and methanol production in bacterial methylotrophy. We mutated decameric Bacillus methanolicus MDH by directed evolution and screened mutants for increased formaldehyde reduction activity in Escherichia coli. The mutant with the highest formaldehyde reduction activity had three amino acid substitutions: F213V, F289L, and F356S. To identify the individual contributions of these residues to the increased reduction activity, the activities of mutant variants were evaluated. F213V/F289L and F213V/F289L/F356S showed 25.3- and 52.8-fold higher catalytic efficiency (kcat/Km) than wild type MDH, respectively. In addition, they converted 5.9- and 6.4-fold more formaldehyde to methanol in vitro than the wild type enzyme. Computational modelling revealed that the three substituted residues were located at MDH oligomerization interfaces, and may influence oligomerization stability: F213V aids in dimer formation, and F289L and F356S in decamer formation. The substitutions may stabilise oligomerization, thereby increasing the formaldehyde reduction activity of MDH.

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