The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes

Abstract

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production.Abbreviations 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; AcK, acetate kinase; ALDO, fructose 1,6-bisphosphate aldolase; CODH/ACS, carbon monoxide dehydrogenase/acetyl-CoA synthetase complex; DHAP, dihydroxyacetone phosphate; Eh-, recombinant enzyme of Entamoeba histolytica; EMP, Embden-Meyerhoff-Parnas; ENO, enolase; Erythro4P, erythrose 4-phosphate; Fru1,6BP, fructose 1,6-bisphosphate; Fru6P, fructose 6-phosphate; FruBPase, fructose 1,6-bisphosphatase; G3P, glyceraldehyde 3-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GAPN, nonphosphorylating GAPDH; GAPOR, glyceraldehyde 3-phosphate: ferredoxin oxidoreductase; Glc1P, glucose 1-phosphate; Glc6P, glucose

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases

“…Both cases agree with current theories for the evolution of enzyme catalysis (32) and recently reviewed by Newton et al (33), which propose that changes in the kinetic parameters depend on the function and metabolic context in which the enzyme works. Considering the above, ADP-dependent kinases from Methanococcales are bifunctional enzymes with low catalytic efficiencies, in accordance with the fact that these organisms obtain their energy mainly from methanogenesis, whereas glycolysis/gluconeogenesis participates in glycogen metabolism, which could be an environmental advantage when carbon sources are scarce (34). On the other hand, in Thermococcales, glycolysis would be a major source of energy, in accordance with the presence of ADP-dependent specific enzymes with higher catalytic efficiencies.…”

Metabolic engineering of Methanosarcina acetivorans for lactate production from methane

“…Por catalyzes the interconversion between acetyl‐CoA and pyruvate (Fig. ), principally in the direction of pyruvate synthesis in vivo (Santiago‐Martinez et al, ), so Por was included in the hopes of increasing lactate production. Analyzing the transformants grown in HSYE with 125 mM methanol by HPLC revealed the production of lactate only in cells producing Hbd (pES1‐MAT mcr 3‐Pfer‐ hbd , pES1‐MAT mcr 3‐P­fer‐ hbd‐por GDAB, and pES1MATbiohol‐B4; Figs.…”

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

“…The model biomass equation was refined by incorporating osmolytes, salts, and metals [61] and gluconeogenesis intermediates [62]. Additionally, pyrrolysine was added as a requirement for growth on methylamines, allowing for the correct prediction of pyl gene knockouts when growing on these substrates (Additional file 1: Figure S11).…”