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.

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.

The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C–C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be subjected to high-throughput evolution studies on account of the lack of an efficient analytical tool for C1 metabolites. To address this challenging issue, we attempted to establish a fine-tuned single-cell–level biosensor system constituting a combination of transcription factors (TFs) and several C1-converting enzymes that convert target compounds to the ligand of a TF. This enzymatic conversion broadens the detection range of ligands by the genetic biosensor systems. In this study, we presented new genetic enzyme screening systems (GESSs) to detect formate, formaldehyde, and methanol from specific enzyme activities and pathways, named FA-GESS, Frm-GESS, and MeOH-GESS, respectively. All the biosensors displayed linear responses to their respective C1 molecules, namely, formate (1.0–250 mM), formaldehyde (1.0–50 μM), and methanol (5–400 mM), and they did so with high specificity. Consequently, the helper enzymes, including formaldehyde dehydrogenase and methanol dehydrogenase, were successfully combined to constitute new versatile combinations of the C1-biosensors.