Methane valorization: construction and validation of whole genome metabolic model of Methylomicrobium alcaliphilum 20Z

Methanotrophs are a very attractive system for valorization of small, scattered and often remote resources of methane. The conversion of methane into targeted value added compounds requires alteration of metabolic pathways. In this study we constructed the flux balance model of the methane utilization network in Methylomicrobium alcaliphilum 20Z using Cobra toolbox. All central metabolic pathways and pathways essential for methane oxidation and assimilation were manually reconstructed. The model consists of three compartments (extracellular space, periplasm and cytoplasm) and includes 385 internal reactions and 17 exchange rates. The model was used to test different routs of methane oxidation, methanol oxidation and make prediction for the most optimal cultivation conditions. To further test the model predictions we performed a set of cultivation and mutagenesis studies. Our data suggest that electrons from methanol oxidation support methane oxidation, however a relatively large part of NADH also enters the respiratory chain, possibly to support an uphill electron transfer. The cytochrome bc1 (ΔpetABC) knockouts showed some growth defects, but were able to utilize methane as its sole source of carbon and energy. The data suggest that cytochrome bc1 is essential for respiration rather than methane oxidation, or, if there is a reversible redox coupling, yet unknown systems must substitute for bc1 complex. A set of novel mutants (Δcox and Δbhr) had wild type methane consumption and displayed reduced oxygen consumption rates, suggesting that methane oxidation is not always linked to oxidative phosphorylation. The mutants are capable of producing organic acids from methane at high concentrations of dissolved oxygen and thus represent novel traits for enhanced fermentation of methane.