Erforschung des Kohlenhydratstoffwechsels in hyperthermophilen Archaeen: Neue Ansätze, Enzyme und Stoffwechselwege
Biochemie
Biologische und Biomimetische Chemie
Mikrobielle Ökologie und Angewandte Mikrobiologie
Zusammenfassung der Projektergebnisse
Archaea, the third domain of life, harbour unique biological traits, including a metabolism that on the one hand shares bacterial and eukaryotic properties but on the second hand also features unique pathways and enzymes. Among them, (hyper)thermophilic archaea are particular interesting as their adaption to extreme environmental conditions (temperature, pH, etc.) has triggered the evolution of a particular enzyme repertoire that can serve as an intriguing source for the discovery of novel enzymes for biotechnological applications (accordingly often referred to as ‘extremozymes’). A broader use of enzymes from (hyper)thermophilic archaea is however currently hampered by a lack of suitable methodologies for their identification and systematic study. Within this project, we therefore aimed to establish alternative experimental methodologies to investigate hyperthermophilic archaeal enzymes, in particular related to carbohydrate metabolism, directly from environmental samples. This was achieved by extending activitybased protein profiling (ABPP), a chemical proteomics technique widely used in the field of (bio)medicine research, from an application to cultured isolated archaeal organisms to whole microbial communities. ABPP allows to detect and identify active enzymes under various physiological conditions via the application of activity-based probes (ABPs) that react in a specific and often enzyme class-specific manner with target enzymes. Although rarely used in normal ABPP workflows, the application of enzyme class-specific ABP may thus allow to identify new enzymes, including even enzymes with so far unannotated functions. This alternative workflow has been made possible by combining ABPP with metagenomic analyses of community samples as well as the implementation of customized workflows for sample workup for mass spectrometry-based ABPP. This alternative ABPP approach has been named by us as ‘environmental ABPP’ (eABPP) and we have showcased its applicability by two ‘environmental’ applications. To this end, either enrichment (grown in presence of different carbohydrate substrates) as well as environmental community probes from microbial sediments were sampled at hot springs in Kamchatka (Russia) and analysed by our ABPP procedures. In the case of the enrichment cultures, the usage of glycosidase ABPs allowed to identify various active glycoside hydrolases from which a thermostable β-glucosidase was further biochemically validated. For the more complex experimental setting, i.e. the eABPP of a sampled sedimentary microbial community, fluorophosphonate-based ABPs were to identify various serine hydrolases directly from these environmental samples, from which an esterase was selected for further biochemical validation. Besides its usage for identifying new enzymes from environmental samples, we further showed that ABPP may also be applied to investigate biochemical pathways in isolated, cultured archaea as demonstrated by an ABPP profiling of the glycosidase repertoire of an isolated Thermococcus sp. strain 2319x1E.
Projektbezogene Publikationen (Auswahl)
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Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E. Frontiers in Microbiology, 12.
Klaus, Thomas; Ninck, Sabrina; Albersmeier, Andreas; Busche, Tobias; Wibberg, Daniel; Jiang, Jianbing; Elcheninov, Alexander G.; Zayulina, Kseniya S.; Kaschani, Farnusch; Bräsen, Christopher; Overkleeft, Herman S.; Kalinowski, Jörn; Kublanov, Ilya V.; Kaiser, Markus & Siebers, Bettina
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Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities. Cold Spring Harbor Laboratory.
Ninck, Sabrina; Klaus, Thomas; Kochetkova, Tatiana V.; Esser, Sarah P.; Sewald, Leonard; Kaschani, Farnusch; Bräsen, Christopher; Probst, Alexander J.; Kublanov, Ilya V.; Siebers, Bettina & Kaiser, Markus
