Recent studies demonstrated that the methanogen Methermicoccus shengliensis is capable of methane production from methoxylated aromatic compounds (MACs), which are released upon degradation of the abundant biopolymer lignin [1, 2]. A bacteria-like corrinoid dependent O-demethylase system (MtoABCD) enables such "methoxydotrophic" lifestyle [2]. Although resembling methyltransferases from conventional methylotrophic methanogens, a key difference is that tetrahydromethanopterin (H4MPT) acts as the terminal CH3-acceptor instead of coenzyme M (CoM). This likely results in an altered energy metabolism and redox (im)balance [2]. Interestingly, two gene isoforms encoding the first methyltransferase that cleaves the O-CH3 bond (mtoB1 and mtoB2) are present in the genome of M. shengliensis, suggesting that these might have different substrate ranges to cover a wider range of metabolizable substrates [2]. Pioneering research showed that methane yields of grown M. shengliensis cultures indeed depend on the type of MAC added to the medium, as well as on the strain [1]. To further elaborate on this, we will combine in vivo and in vitro experiments: 1) For a selection of substrates, we will perform growth experiments while following production of methane (GC measurements). RT-qPCR should reveal upregulation of either of the mtoB genes in response to a specific substrate; 2) MtoB1/MtoB2 will either be natively purified from M. shengliensis based on RT-qPCR information to specifically enrich either of the isoforms. Moreover, research is ongoing to heterologously produce these two MtoB isoforms. Preliminary data obtained so far revealed that Escherichia coli does not properly produce MtoB, but the reason for this issue remains yet to be elucidated. In conclusion, an integration of in vivo and in vitro approaches should provide us with novel insights regarding substrate specificities and preferences of the Mto-system from M. shenglienis.

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

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