Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Neodymium as Metal Cofactor for Biological Methanol Oxidation: Structure and Kinetics of an XoxF1-Type Methanol Dehydrogenase

Lanthanides comprise a group of 15 elements with atomic numbers 57 to 71 that are essential in a variety of high-tech devices, such as mobile phones, but were considered biologically inert for a long time. The biological relevance of lanthanides became evident when the acidophilic methanotroph Methylacidiphilum fumariolicum SolV, isolated from a volcanic mud pot, could only grow when lanthanides were supplied to the growth medium.

Switching Between Methanol Accumulation and Cell Growth by Expression Control of Methanol Dehydrogenase in Methylosinus trichosporium OB3b Mutant

Methanotrophs have been used to convert methane to methanol at ambient temperature and pressure. In order to accumulate methanol using methanotrophs, methanol dehydrogenase (MDH) must be downregulated as it consumes methanol. Here, we describe a methanol production system wherein MDH expression is controlled by using methanotroph mutants. We used the MxaF knockout mutant of Methylosinus trichosporium OB3b. It could only grow with MDH (XoxF) which has a cerium ion in its active site and is only expressed by bacteria in media containing cerium ions. In the presence of 0 μM copper ion and 25 μM cerium ion, the mutant grew normally. Under conditions conducive to methanol production (10 μM copper ion and 0 μM cerium ion), cell growth was inhibited and methanol accumulated (2.6 μmol·mg−1 dry cell weight·h−1). The conversion efficiency of the accumulated methanol to the total amount of methane added to the reaction system was ~0.3%. The aforementioned conditions were repeatedly alternated by modulating the metal ion composition of the bacterial growth medium.

A Novel Moderately Thermophilic Facultative Methylotroph within the Class Alphaproteobacteria

Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon–carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.

Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus

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.

Methanol Production by “Methylacidiphilum fumariolicum” SolV under Different Growth Conditions

ABSTRACT Industrial methanol production converts methane from natural gas into methanol through a multistep chemical process. Biological methane-to-methanol conversion under moderate conditions and using biogas would be more environmentally friendly. Methanotrophs, bacteria that use methane as an energy source, convert methane into methanol in a single step catalyzed by the enzyme methane monooxygenase, but inhibition of methanol dehydrogenase, which catalyzes the subsequent conversion of methanol into formaldehyde, is a major challenge. In this study, we used the thermoacidophilic methanotroph “Methylacidiphilum fumariolicum” SolV for biological methanol production. This bacterium possesses a XoxF-type methanol dehydrogenase that is dependent on rare earth elements for activity. By using a cultivation medium nearly devoid of lanthanides, we reduced methanol dehydrogenase activity and obtained a continuous methanol-producing microbial culture. The methanol production rate and conversion efficiency were growth-rate dependent. A maximal conversion efficiency of 63% mol methanol produced per mol methane consumed was obtained at a relatively high growth rate, with a methanol production rate of 0.88 mmol/g (dry weight)/h. This study demonstrates that methanotrophs can be used for continuous methanol production. Full-scale application will require additional increases in the titer, production rate, and efficiency, which can be achieved by further decreasing the lanthanide concentration through the use of increased biomass concentrations and novel reactor designs to supply sufficient gases, including methane, oxygen, and hydrogen. IMPORTANCE The production of methanol, an important chemical, is completely dependent on natural gas. The current multistep chemical process uses high temperature and pressure to convert methane in natural gas to methanol. In this study, we used the methanotroph “Methylacidiphilum fumariolicum” SolV to achieve continuous methanol production from methane as the substrate. The production rate was highly dependent on the growth rate of this microorganism, and high conversion efficiencies were obtained. Using microorganisms for the production of methanol might enable the use of more sustainable sources of methane, such as biogas, rather than natural gas.