Tracing the gaps - Unraveling initial steps in hexose metabolism in Saccharolobus solfataricus using an improved stable isotope labeling approach

Archaea are often found in harsh habitats withstanding for instance high temperatures, salinity and acidic or alkaline environments making them valuable models to understand life under extreme conditions. Their metabolism is characterized by unique and unusual metabolic pathways and enzymes that differ from bacteria and eukaryotes. Among the Thermoproteota, Saccharolobus solfataricus represents an important model organism for archaeal metabolism. It thrives under thermoacidophilic conditions with an optimum temperature of 80 °C and a pH value between 2 and 4. Additionally, it is characterized by its metabolic versatility, which allows it to grow on a wide variety of 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 instead of the Embden-Meyerhof-Parnas (EMP) pathway commonly used by other organisms. However, the degradation of other hexoses, such as D-mannose and D-fructose, is poorly understood so far. An initial ¹³C₆-mannose spiking experiment indicated that the ED pathway is involved in D-mannose degradation. Due to the absence of homologues enzymes for known D-fructose degradation, a completely novel pathway is hypothesized. To unravel these metabolic blind spots, a GC-MS-based method has been optimized to intentionally slow down the metabolism of S. solfataricus. In this way, the metabolic flux of ¹³C₆-labeled D-mannose and D-fructose can be traced in the first steps of degradation, paving the way for future elucidation and mapping of the degradation pathway.