MetaboArchaea - D-Mannose and D-fructose utilization in members of the Sulfolobales – Significance of the upper Embden-Meyerhof-Parnas pathway

• Applicants: Privatdozentin Dr. Meina Neumann-Schaal; Professorin Dr. Bettina Siebers
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 509500657

DMSZ BraunschweigThe complexity of the archaeal metabolism resembles those of Bacteria and lower Eukaryotes, however, even classical generic pathways involved in central carbohydrate metabolism are characterized by many novel pathways and enzymes that show no similarity to the respective bacterial and eukaryotic counterparts. The utilization of novel enzymes, often from different enzyme families, is accompanied by novel enzymatic and regulatory properties with a major impact on metabolic regulation. The model systems of this project are the thermoacidophilic Crenarchaeota Sulfolobus (now Saccharolobus) solfataricus P2 and Sulfolobus acidocaldarius DSM 639. For Sulfolobus spp. a wealth of biochemical and genetic information is available and recently, knowledge on the metabolic versatility was improved by polyomics and systems biology approaches. S. solfataricus has been reported to grow aerobically on numerous different carbon sources such as sugars, alcohols and amino acids. The pathways for D-glucose/galactose and pentoses degradation via the promiscuous branched Entner-Doudoroff pathway and the Weimberg or Dahms pathway, respectively, are well established. Although S. solfataricus grows on D-mannose and D-fructose, the degradation pathways are unknown. In most microorganisms, they are degraded via the Embden-Meyerhof-Parnas (EMP) pathway. However, this pathway is only used for anabolism in S. solfataricus due to a missing phosphofructokinase suggesting novel pathways for D-mannose and D-fructose degradation. Initial 13C6-D-Mannose spiking experiments point to the presence of a modified ED pathway, the upper EMP pathway as well as alternative metabolic routes but the respective pathways and enzymes still need to be identified and the respective enzymes characterized.In contrast to S. solfataricus, S. acidocaldarius is not able to grow on D-mannose or D-fructose as sole carbon source. However, D-mannose is a major constituent in the tri-branched glucan tree of the S-layer and in the extracellular polymeric substances (EPS) in S. acidocaldarius. Therefore, D-mannose plays also an essential role in archaeal N- and O-glycosylation and EPS formation in both organisms.The aim of this study is to unravel the pathways for degradation of D-mannose and D-fructose in S. solfataricus and to identify and characterize the involved enzymes as well as their regulatory properties by using transcriptome, proteome and metabolome studies, high resolution mass spectrometry as well as crude extract measurements and the characterization of the respective recombinant enzymes. The significance as well as the pathway for D-mannose and UDP-mannose formation will be addressed in S. acidocaldarius by the construction of deletion strains and recombinant enzyme characterization. The aim is to unravel the physiological function of this pathway.

• DFG Programme: Research Grants