Discovery and Biochemical Characterization of a Methanol Dehydrogenase From Lysinibacillus xylanilyticus

Abstract

Bioconversion of C1 chemicals such as methane and methanol into higher carbonchain chemicals has been widely studied. Methanol oxidation catalyzed by methanol dehydrogenase (Mdh) is one of the key steps in methanol utilization in bacterial methylotrophy. In bacteria, few NAD +-dependent Mdhs have been reported that convert methanol to formaldehyde. In this study, an uncharacterized Mdh gene from Lysinibacillus xylanilyticus (Lxmdh) was cloned and expressed in Escherichia coli. The maximum alcohol oxidation activity of the recombinant enzyme was observed at pH 9.5 and 55 • C in the presence of 10 mM Mg 2+. To improve oxidation activity, rational approach-based, site-directed mutagenesis of 16 residues in the putative active site and NAD +-binding region was performed. The mutations S101V, T141S, and A164F improved the enzyme's specific activity toward methanol compared to that of the wild-type enzyme. These mutants show a slightly higher turnover rate than that of wild-type, although their K M values were increased compared to that of wild-type. Consequently, according the kinetic results, S101, T141, and A164 positions may related to the catalytic activity in the active site for methanol dehydrogenation. It should be further studied other mutant variants with high activity for methanol. In conclusion, we characterized a new Lxmdh and its variants that may be potentially useful for the development of synthetic methylotrophy in the future.

Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus

“…The wild-type Lxmdh has been reported to demonstrate relatively high substrate specificity toward methanol when compared with Mdh enzymes from C. necator , B. methanolicus , and B. stearothermophilus [ 4 , 7 , 8 ]. The reported K M value of Lxmdh for methanol was 3.23 mM [ 8 ].…”

“…The wild-type Lxmdh has been reported to demonstrate relatively high substrate specificity toward methanol when compared with Mdh enzymes from C. necator , B. methanolicus , and B. stearothermophilus [ 4 , 7 , 8 ]. The reported K M value of Lxmdh for methanol was 3.23 mM [ 8 ]. This value was quite low compared to that of Mdh and Mdh2 derived from B. methanolicus (9 mM) [ 20 ], Mdh from C. necator (132 mM) [ 4 ] and Mdh, Mdh2, and Mdh3 from B. methanolicus (170–360 mM) [ 7 ].…”

“…E. coli DH5α was used as a cloning host and for the expression of Lxmdh. The recombinant vector for constitutive Lxmdh expression was constructed using pET28a/Lxmdh as a template, which was cloned in a previous study [ 8 ]. The T7 promoter of pET28a/Lxmdh was replaced with the trc promoter to apply E. coli DH5α.…”

“…In a previous study, we developed a new ACT-independent Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has high activity. Additionally, rational approach-based mutation studies resulted in mutants with higher turnover rate compared to the wild-type, even though the K M values were comparatively increased [ 8 ]. However, the improvement of Lxmdh catalytic activity and substrate affinity toward low-concentration methanol remains a challenge in the development of Mdh-driven synthetic methylotrophs.…”

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy

“…(such as B. methanolicus (Arfman et al, 1989;Arfman et al, 1991;Müller et al, 2015;Witthoff et al, 2015;Price et al, 2016) and B. stearothermophilus (Whitaker et al, 2017)), Lysinibacillus sp. (such as L. xylanilyticus (Lee et al, 2020)), and Cupriavidus sp. (such as C. necator (Wu et al, 2016)).…”