Unraveling the functional relevance of two O-demethylase paralogs encoded by Methermicoccus shengliensis

Although methanogenic archaea were originally thought to exclusively feed on small metabolites released from microbial fermentation processed in anaerobic environments, it was demonstrated recently that the methanogen Methermicoccus shengliensis can produce methane from methoxylated aromatic compounds (MACs) [1]. Such compounds are readily released upon depolymerization of the abundant plant biopolymer lignin. It was discovered that the methyltransferase system involved in the demethylation of MACs (MtoABCD) is homologous to such systems of acetogenic bacteria [2]. In contrast to conventional methylotrophic methanogens, this enzyme system feeds the methyl moiety into the methanogenesis pathway using tetrahydromethanopterin (H4MPT) as a cofactor, rather than coenzyme M (CoM) [2]. The mto operon contains two copies of the O-demethylating methyltransferase (mtoB1 and mtoB2), and it has been proposed that each of the paralogs has different substrate preferences [2]. To investigate the substrate usage, we have cultivated M. shengliensis with about 30 different MACs as substrates, but only a minority of these substrates can be used for growth. To understand if this finding is caused by a limitation of the substrate range of the two O-demethylases or by other effects such as toxicity or regulatory issues we will also perform in vitro activity assays using UV-Vis spectroscopy to compare reaction kinetics of MtoB1 and MtoB2 for different substrates. For the cultures that can be grown to feasible cell densities, an RT-qPCR based experiment was designed to compare transcription of mtoB1 and mtoB2 in response to the presence of different MACs. In conclusion, a combination of in vivo and in vitro work provides us with insights in the MAC preferences of M. shengliensis and potential differential regulation of the two encoded MtoB paralogs in response to MAC availability. Moreover, this project should shed light on the presumed specialization of the two MtoB homologs to demethylate different MACs based on kinetic insights.

[1] Mayumi et al. (2016) Science 354: 222

[2] Kurth et al. (2021) ISME J 15: 3549