Physiological barriers for glucose utilization in Methanosarcina acetivorans

Methanogenesis is a key aspect of anaerobic biomass degradation, and thus of global importance. While methanogenic archaea (methanogens) are ubiquitous in anaerobic habitats, the range of substrates they utilize is very limited. Most methanogens are able to grow chemolithotrophically with H2+CO2, some clades can utilize methylated compounds and/or acetate as well. Organotrophic compounds like amino acids, lipids, nucleotides, or carbohydrates, are not known to support growth of methanogens. This inability to utilize "upstream" intermediates of anaerobic biomass degradation is remarkable considering the presence of cellular metabolism that such intermediates could feed into. Here, we addressed the question why the model methanogen Methanosarcina acetivorans, despite its gluconeogenic and glycolytic capacity, is unable to utilize glucose for methanogenesis and growth. Complementing heterologously with a glucose uptake facilitator allowed a recombinant M. acetivorans strain to convert glucose to methane at a low rate. However, growth with glucose was not observed, neither as energy source nor as carbon source. Instead, methylotrophic growth of the transgenic strain was impaired in a glucose-dependent fashion, which was aggravated when also glucokinase was heterologuously produced. Glucose-dependent growth inhibition coincided with a significant - and microscopically visible - accumulation of intracellular carbohydrate. Since the glucose-utilizing trait is rapidly lost during methylotrophic growth, accumulating growth-inhibiting metabolites probably makes methanogenic and glycolytic catabolism incompatible. Thus, extensive efforts in strain development would be required to enable direct glucose utilization for methanogenesis. ImportanceThe known range of methanogenic growth substrates is very limited and the most reduced carbon compound to support organotrophic growth of M. acetivorans is pyruvate. Yet, gluconeogenesis and glycolysis, i. e., mobilization of stored glycogen, which is known to occur in M. acetivorans makes the capacity of glycolytic methanogenesis in this organism seem feasible. Although this trait could be engineered in M. acetivorans by introducing a single gene, the finding that it could not sustain growth of the organism, not even as an anabolic carbon source, demonstrates that methanogenesis is not easily compatible with carbohydrate utilization, thus probably requiring extensive metabolic rewiring.