Mutagenesis of the C1 oxidation pathway in Methanosarcina barkeri:: New insights into the Mtr/Mer bypass pathway

A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin (H4MPT):coenzyme M (CoM) methyltransferase-encoding operon (Delta mtr), the methylene-H4MPT reductase-encoding gene (Delta mer), the methylene-H4MPT dehydrogenase-encoding gene (Delta mtd), and the formyl-methanofuran:H4MPT formyl-transferase-encoding gene (Delta ftr) all failed to grow using either methanol or H-2/CO2 as a growth substrate, indicating that there is an absolute requirement for the Cl oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the Delta mtr and Delta mer mutants were capable of producing methane from this substrate, whereas the Delta mtd and Delta ftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-H4MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in Delta ntr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and H4MPT in vitro. Both the Delta mer and Delta mtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.