Targeting methanol formation for reducing methane emissions

Pectin is a complex polysaccharide that forms a substantial proportion of the plant cell wall of forage ingested by grazing ruminants. Methanol derives from methoxy groups released from pectin by the action of pectin methylesterases (PMEs), and is subsequently used by rumen methylotrophic methanogens, such as members of the genus Methanosphaera, that reduce methyl-compounds to methane (CH₄). Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation as well as having important roles in fibre breakdown, protein digestion and biohydrogenation of fatty acids. The aim of this study is to investigate pectin breakdown in Butyrivibrio and to find ways to inhibit these activities to reduce methanol release from pectin. Comparative genomics identified candidate Butyrivibrio strains containing genes encoding pectin degradation and methanol release. The growth of Butyrivibrio strains was tested on high- and low-methoxylated pectin and their fermentation products were analysed to confirm their predicted pectin-degrading (pec^+/-) and methanol-forming (MeOH^+/-) phenotypes. To investigate metabolic coupling of high methanol producing Butyrivibrio strains with rumen methanogens, we performed a time-series RNA-seq experiment on co-cultures of pec⁺/MeOH⁺ B. proteoclasticus B316^T and B. fibrisolvens D1^T with a methylotrophic methanogen Methanosphaera sp. ISO3-F5 grown on pectin. Structures of selected Butyrivibrio PMEs have been modelled with bound substrate to identify the amino acid residues best suited for inhibitor binding and inhibitor library screening. With selected compounds synthesized for upcoming in vitro screening, this strategy represents an alternative approach to methane mitigation which currently inhibit methanogenesis directly.