Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications

Abstract

Worldwide, the use of methane is limited to generating power, electricity, heating, and for production of chemicals. We believe this valuable gas can be employed more widely. Here we review the possibility of using methane as a feedstock for biotechnological processes based on the application of synthetic methanotrophs. Methane monooxygenase (MMO) enables aerobic methanotrophs to utilize methane as a sole carbon and energy source, in contrast to industrial microorganisms that grow on carbon sources, such as sugar cane, which directly compete with the food market. However, naturally occurring methanotrophs have proven to be difficult to manipulate genetically and their current industrial use is limited to generating animal feed biomass. Shifting the focus from genetic engineering of methanotrophs, towards introducing metabolic pathways for methane utilization in familiar industrial microorganisms, may lead to construction of efficient and economically feasible microbial cell factories. The applications of a technology for MMO production are not limited to methane-based industrial synthesis of fuels and value-added products, but are also of interest in bioremediation where mitigating anthropogenic pollution is an increasingly relevant issue. Published research on successful functional expression of MMO does not exist, but several attempts provide promising future perspectives and a few recent patents indicate that there is an ongoing research in this field. Combining the knowledge on genetics and metabolism of methanotrophy with tools for functional heterologous expression of MMO-encoding genes in non-methanotrophic bacterial species, is a key step for construction of synthetic methanotrophs that holds a great biotechnological potential.

Keywords: Bioprocesses; Bioremediation; Gas-to-liquid; Methane monooxygenase; Synthetic methanotrophy.

Fig. 1

Metabolic pathways of methanotrophs type I, II and X. Methane (CH₄) is oxidized to methanol (CH₃OH) by action of soluble methane monooxygenase (sMMO) or particulate methane monooxygenase (pMMO). Methanol dehydrogenase (MDH) then converts methanol to formaldehyde (HCHO). Type I and X methanotrophs utilize the RuMP pathway (orange) to assimilate formaldehyde to biomass. Type II methanotrophs utilize the serine pathway (green) for formaldehyde assimilation. All types of methanotrophs also have formaldehyde dissimilation pathways (middle). Full arrows indicate a single process and dashed arrows indicate several reactions. Abbreviations: RuMP, ribulose monophosphate; H6P, hexulose-6-phosphate; F6P, fructose-6-phosphate; Ru5P, ribulose-5-phosphate; H4F, tetrahydrofolate; malyl-CoA, malyl co-enzyme A

Fig. 2

sMMO- and pMMO-encoding operons of M. capsulatus (Bath) and of M. trichosporium OB3b. Six genes (yellow) encode sMMO. mmoX, mmoY and mmoZ encode respectively the α-, β- and γ-subunit of the hydroxylase MMOH. mmoB encodes the regulator protein MMOB. mmoD encodes a disputed regulatory protein MMOD. mmoC encodes the reductase MMOC. mmoG (blue) encodes MMOG, a GroEL-like chaperonin; mmoQ and mmoS (orange) encode a two-component sensor system believed to be responsible for the copper-switch; and mmoR (green) encodes a reductase MMOR. Hypothetical protein / unidentified open reading frame (grey). Three genes pmoC, pmoA and pmoB (purple) encode the three subunits of pMMO, PmoA, PmoB and PmoC, respectively. Arrows show promoter sites and indicate the family of sigma factors associated with them (Cardy et al. ; Holmes et al. ; Ward et al. ; Lieberman and Rosenzweig 2004)