Stable isotope labeling uncovers novel hexose degradation routes in Saccharolobus solfataricus

Archaea are often found in extreme environments, tolerating for instance high temperatures, salinity and acidic or alkaline environments making them valuable models for understanding life under such conditions. Their metabolism is often characterized by unique and unusual metabolic pathways and enzymes that differ from bacteria and eukaryotes. Within the Thermoproteota, Saccharolobus solfataricus is a key model organism for studying archaeal metabolism. It thrives under thermoacidophilic conditions with an optimum temperature of 80 °C and a pH range of 2 to 4. Additionally, S. solfataricus is characterized by its metabolic versatility, allowing growth on various carbon sources. For some hexoses like D-glucose and D-galactose the central carbohydrate metabolism has been extensively investigated, revealing a modified branched Entner-Doudoroff (ED) pathway for degradation. This pathway notably avoids phosphorylated intermediates, which are common in other organisms. However, the degradation of other hexoses, such as D-mannose and D-fructose, remain unexplored. To address these metabolic gaps, a stable isotope labeling experiment was optimized to enable time-resolved tracing of D-mannose and D-fructose degradation. The labeling experiments suggest degradation analogous to the modified ED pathway, but with a surprisingly high flux through intermediates like mannose-6-phosphate, fructose-6-phosphate, and trehalose, indicating a so far unknown degradation route within the S. solfataricus catabolism.