Overexpression of 2-mercaptoethanesulfonate biosynthesis genes comDE protects methane-producing archaea from oxidative stress.

UNLABELLED: Coenzyme M (2-mercaptoethane sulfonate, CoM) is an essential low-molecular-weight thiol in methanogenic archaea (methanogens) that serves as a methyl carrier and as a component of CoM-S-S-CoB heterodisulfide comprising CoM and coenzyme B (7-mercaptoheptanoylthreoninephosphate), which serves as the terminal electron acceptor in methanogenesis. Increasing the amount of CoM in Methanosarcina acetivorans cells by overexpressing its proposed biosynthesis genes results in faster growth on methanol or methanol + acetate medium in the absence of sulfide. Furthermore, CoM overproduction enhances resistance to oxidative stress in the ΔhdrABC mutant genetic background in which the nonessential methylotrophic-specific CoM-S-S-CoB heterodisulfide reductase enzyme, HdrABC, has been deleted. The ΔhdrABC mutant is resistant to 5% O2 atmosphere, and overexpression of CoM biosynthesis genes resulted in resistance to up to 2 mM hydrogen peroxide in a stress assay. Increased resistance to O2 is correlated with 34% lower CoM-SH level in the ΔhdrABC and 16% lower CoM-SH level in the ΔhdrABC com+ mutant strains versus the parent strain, while resistance to H2O2 stress is correlated to a higher ratio of reduced CoM-SH to total CoM in the parent versus the com+ (8.7%), ΔhdrABC (10%), and ΔhdrABC com+ (16%) mutant strains. Our study suggests increased expressions of genes encoding coenzyme M biosynthesis, in conjunction with deletion of HdrABC, increase oxidative stress resistance of the strictly anaerobic methanogen Methanosarcina acetivorans.

IMPORTANCE: Methanogens are key organisms in the global carbon cycle with potential to be harnessed to produce renewable energy and transportation fuel. Methanogens are strict anaerobes found in subsurface sediment, anaerobic digesters, and digestive tracts of animals such as the rumen. Our results suggest methanogens can adapt to prolonged exposure to oxidative stress under the appropriate environmental conditions, and it may be possible to engineer thiol redox homeostasis and oxidative stress resistance in methanogens. Engineering redox homeostasis and oxidative stress resistance in methanogens and other strict anaerobes has the potential to reduce technical barriers to culturing, thus accelerating research progress on a wide variety of non-model microbes, and ultimately broadening potential biotechnology applications related to sustainable food, fuel, and biomedical uses.