Reconstruction of a Genome Scale Metabolic Model of the polyhydroxybutyrate producing methanotroph Methylocystis parvus OBBP

Abstract

Background: Methylocystis parvus is a type II methanotroph characterized by its high specific methane degradation rate (compared to other methanotrophs of the same family) and its ability to accumulate up to 50% of its biomass in the form of poly-3-hydroxybutyrate (PHB) under nitrogen limiting conditions. This makes it a very promising cell factory.

Results: This article reports the first Genome Scale Metabolic Model of M. parvus OBBP. The model is compared to Genome Scale Metabolic Models of the closely related methanotrophs Methylocystis hirsuta and Methylocystis sp. SC2. Using the reconstructed model, it was possible to predict the biomass yield of M. parvus on methane. The prediction was consistent with the observed experimental yield, under the assumption of the so called "redox arm mechanism" for methane oxidation. The co-consumption of stored PHB and methane was also modeled, leading to accurate predictions of biomass yields and oxygen consumption rates and revealing an anaplerotic role of PHB degradation. Finally, the model revealed that anoxic PHB consumption has to be coupled to denitrification, as no fermentation of PHB is allowed by the reconstructed metabolic model.

Conclusions: The "redox arm" mechanism appears to be a general characteristic of type II methanotrophs, versus type I methanotrophs that use the "direct coupling" mechanism. The co-consumption of stored PHB and methane was predicted to play an anaplerotic role replenishing the serine cycle with glyoxylate and the TCA cycle with succinyl-CoA, which allows the withdrawal of metabolic precursors for biosynthesis. The stored PHB can be also used as an energy source under anoxic conditions when coupled to denitrification.

Keywords: Genome-scale metabolic models; Metabolism; Methanotrophs; Methylocystis.

Fig. 1

Venn diagrams representing the shared and unique reactions (a) and metabolites (b) of the GSMMs of Methylocystis parvus OBBP, Methylocystis hirsuta and Methylocystis sp. SC2. c The logarithms of the numbers (n) of metabolites participating in k reactions. The plot shows a typical scale free topology. d The same plot including only reactions that are active under optimal growth on methane, and their metabolites

Fig. 2

Schematic representation of the three possible methane oxidation mechanisms: “redox arm” (a), “uphill electron transfer” (b) and “direct coupling” (c). Comparison of the biomass yields on methane predicted by the model under each mechanism with the experimental yield observed. d Error bars correspond to standard deviations from triplicate experiments. M. parvus OBBP biomass produced as a function of methane degraded (e)

Fig. 3

Schematic representation of the metabolic processes active during PHB and methane co-consumption (a). Methane and oxygen consumption per gram of produced biomass during PHB co-consumption with methane (b). The experimental yields of methane and oxygen consumed per unit of synthesized biomass are compared to the yields predicted by the GSMM model

Fig. 4

Predicted maximal ATP produced and nitrate consumed per mol of degraded PHB versus secreted acetate (a). Predicted maximal ATP produced and nitrate consumed per mol of degraded PHB versus secreted butane-2,3-diol (b). Predicted maximal butane-2,3-diol produced and nitrate consumed per mol of degraded PHB versus secreted acetate (c)

Fig. 5

Structure of the gene clusters of particulate methane monooxygenases present in M. parvus, M. hirsuta and M. sp. SC2 (a). Phylogenetic trees showing the relative sequence similarity of each of the three subunits (b)

Fig. 6

Multiple alignments showing the metallic catalytic sites of pMMO. The dinuclear copper site formed by three histidine residues in subunit B is highlighted in red. The zinc site, formed by two histidine and one aspartic acid residue in subunit C, is highlighted in blue. The mononuclear cooper site, formed by a histidine and an asparagine residue in subunit B, is highlighted in green. In the subunit B3, present in M. hirsuta, asparagine was changed into glutathione