Table 1.

Strategies for expanding the metabolic potential of the methanogenesis pathway.

Desired trait	Potential mechanism
Increased methanogenesis and methane production	Overexpression of genes associated with methanogenesis or addition of parallel heterologous methanogenic pathways. Exogenous addition of metabolites or pathway engineering to supply limiting metabolites. Research has shown that overexpression of redox-active cofactors such as methanophenazine in Methanosarcinales relieves the metabolic bottleneck caused by cofactor regeneration and increases the production of methane (Catlett et al., 2015). Vitamins addition often stimulates growth (Tanner and Wolfe, 1988; Jin et al., 2017). Many methanogens are fully prototrophic, but some strains are dependent on exogenous addition of CoM or other vitamins (Catlett et al., 2022). Additionally, magnetite nanoparticles have been demonstrated to serve facilitate increased acetotrophic methanogenesis in cocultures between acetogens and methanogens (Tanner and Wolfe, 1988; Catlett et al., 2015; López Muñoz et al., 2015; Jin et al., 2017; Fu et al., 2019; Catlett et al., 2022).
Increased substrate uptake rates	By overexpressing endogenous or ortho/heterologous methyltransferases and hydrogenases more substrate carbon could enter methanogenesis. In methylotrophic methanogenesis entry point methanogenesis is limited by the substrate-specific methyltransferase whereas hydrogenotrophic methanogens rely upon membrane bound methyltransferase to conserve energy and maintain the methanogens sodium motive force (Kurth et al., 2020).
Increased substrate diversity and mixotrophy	Introduction of multiple substrate input pathways would allow more rapid substrate uptake, faster unitrophic growth, and mixotrophic growth. Substrate entry into methanogenesis is limited by substrate specific methyltransferases and whether the methanogen can directly utilize H2 as an electron source. By introducing methyltransferases from different methanogens one can expand the substrates usable to the methanogen. Increasing extracellular-facing hydrogenases may allow increased rates of H2 uptake and hydrogenotrophic methanogenesis. Upregulation of pyruvate ferredoxin oxidoreductase (por) in M. barkeri has been demonstrated to facilitate growth on pyruvate as a sole carbon and energy source (López Muñoz et al., 2015).
Controlled energy conservation	Selective uncoupling biomass from methanogenesis could allow maximal growth for bioreactor scale-up with a methanogenesis-only production phase. This could be accomplished by bypassing ATP synthesis, managing macromolecular accessibility, by adding protein synthesis inhibitors, or futile cycling for redox cofactors either chemically or genetically (Catlett et al., 2015).
Increased stress resistance	Increased stress tolerance could increase growth rate, improve expression of introduced enzymes, enable production of xenobiotic chemicals, and expand biorefining process parameters. All methanogens are strict anaerobes. Increased oxygen tolerance was observed in Methanosarcina acetivorans when gradually passaged with increased O2 concentrations over a course of 6 months (Jasso-Chávez et al., 2015). Transcripts from adapted Methanosarcina suggest the over expression of superoxide dismutase, catalase, and peroxidase will confer increased aerotolerance to other methanogens. Methanogens engineered to express the bacterial catalase EcKatG demonstrated increased tolerance of hydrogen peroxide, though no increase in resistance to O2 was observed (Jennings et al., 2014). Though non-spore-forming, methanogens are capable of revival after desiccation with no significant loss of viability observed in aerobic environments (Anderson et al., 2012). Cocultivation with sulfate reducing bacteria has shown to mitigate heavy metal stress in methanogenic cultures (Paulo et al., 2015). The introduction or overexpression of the betaine transporter from Methanosarcina thermophila TM-1 increases internal ionic balance conferring protection against osmotic stress (Macario and Macario, 2003). Additionally, it has been noted that under high ammonia conditions which inhibits acetotrophic methanogenesis, the addition of magnetite reduces inhibition (Macario and Macario, 2003; Anderson et al., 2012; Horne and Lessner, 2013; Jennings et al., 2014; Jasso-Chávez et al., 2015; Paulo et al., 2015; Wang et al., 2020).
Multiple	Trait stacking for process optimization. By stacking the above traits may be possible to maximize methanogenic efficiency in mixed substrate environments such as the treatment of waste biomass or in process conditions that require multiple extremophilic conditions.

Genetic methods in methanoarchaea are available in several species. Current tools have been recently reviewed or published (Tumbula et al., 1994; Pritchett et al., 2004; Ehlers et al., 2005; Guss et al., 2008; Atomi et al., 2012; Kohler and Metcalf, 2012; Nayak and Metcalf, 2017; Innard, 2018).