(Halo)alcaliphilic methanotrophs: new microbial platform for producing fuels and chemicals from methane

The long-term goal of this research is to develop a robust microbial platform for conversion of greenhouse gases (CH₄) into valuable chemicals as an alternate approach to widespread (bio)gas-flaring.  Physiological properties of the (halo)alcaliphilic strains, such as Methylomicrobium alcaliphilum, make them unique targets for the development of green-technologies based on wasted methane. Here we explore the applicability of M. alcaliphilum as a new platform for production of chemicals from methane. 

A stoichiometric model of M. alcaliphilum was constructed in PathwayTools and used for evaluating the metabolic network. The model was used to test different arrangements of methane oxidation (i) redox-arm mode, the currently accepted model in which electrons driving methane oxidation come from NADH produced by formate or formaldehyde oxidation, while electrons produced from methanol oxidation are linked to redox-arm and used for ATP production; and (ii) direct coupling (DC), in which methanol oxidation supplies electrons directly for methane oxidation. Only the latter model predictions correlated well with the experimental growth data. To further validate the DC-model, we mutated the cytochrome oxidase (cox), NADH-oxidoreductase (ndh2) and the cytochrome bc1 complex (petA) genes.  All three mutants grew on methane, confirming that methanol oxidation does not operate as a redox-arm. While still in the development mode the flux balance model was applied to facilitate metabolic engineering of the methanotrophic network in Methylomicrobium spp.  Two sets of mutants with h CH₄-to-pyruvate conversion efficiencies (Y_gC3/Y_gCH4 ) up to 0.23 were constructed.  The succinate production in the strain is being improved via heterologous expression approaches.