PEROXIDIV5 - Phylogenetische und funktionelle Diversität pilzlicher Häm-Peroxidasen (PeroxiDiv)
Biologische und Biomimetische Chemie
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Zusammenfassung der Projektergebnisse
Fungi represent one of the main sources of enzymes used in industry. New peroxidase families (dye-decolorizing peroxidases - DyPs and unspecific peroxygenases - UPOs) from fungi have recently emerged as promising biocatalysts because of their catalytic versatility and potential use in diverse industrial domains, from bioremediation via fine chemistry to the pharmaceutical sector. The objective of PeroxiDiv was to explore the diversity of these biocatalysts using different approaches. On the one hand, datamining of recently sequenced fungal genomes; on the other hand, the development and implementation of an environmental genomics strategy that allows the capture and characterization of novel genes expressed by microorganisms present in complex environments (soils, sediments, decomposing wood). A second part of the project aimed at the production of these new proteins in heterologous microbial hosts to describe their catalytic properties on the basis of different relevant substrates. Analysis of peroxidase diversity necessitates the implementation of bioinformatics and phylogenetic approaches to (i) identify the corresponding genes within public databases and (ii) infer the evolutionary history of these protein families. Then, confrontation between “real” and “known” diversity could be used to identify sequences, to focus on in order to carry out further functional characterization. The characterization of peroxidase genes expressed in environmental samples necessitated the implementation of RNA extraction protocols from decaying wood, soil and sediments. Oligonucleotide probes representative of the studied gene families were designed in order to capture the environmental sequences by hybridization, which were sequenced using highthroughput technologies. The functional characterization of new peroxidases, including those from the environment, necessitated their production as active recombinant proteins in microbial hosts. Production of these families of enzymes is often problematic and therefore, different bacterial and fungal hosts (including yeasts and filamentous fungi) were tested. Fungal DyP and UPO protein families display an unexpected diversity characterized by the occurrence of numerous putatively intracellular enzymes that could display novel catalytic properties. New peroxidases have been captured by hybridization from different environments and their sequences reconstructed for the first time using bioinformatic tools from highthroughput sequence information. Production of more than 20 different enzymes has been attempted in 3 different microbial hosts (one bacterium, one filamentous fungus and one yeast), but resulted in inactive proteins or small amounts of low-activity proteins. In the latter regard, more research will be needed to take full advantage of this environmental approach, i.e. as long as recombinant expression of heme peroxidases along all their limitations are not fully addressed, the panoply of newly identified genes will be futile for White Biotechnology.
Projektbezogene Publikationen (Auswahl)
- 2017. Discovering Protein-Coding Genes from the Environment: Time for the Eukaryotes? Trends in Biotechnology, Special Issue: Environmental Biotechnology 35, 824–835
Marmeisse, R., Kellner, H., Fraissinet-Tachet, L., Luis, P.
(Siehe online unter https://doi.org/10.1016/j.tibtech.2017.02.003) - 2017. Draft Genome Sequence of the Wood-Degrading Ascomycete Kretzschmaria deusta DSM 104547. Genome Announc 5
Büttner, E., Gebauer, A.M., Hofrichter, M., Liers, C., Kellner, H.
(Siehe online unter https://doi.org/10.1128/genomeA.01076-17) - 2018. Draft Genome Sequence of the Sordariomycete Lecythophora (Coniochaeta) hoffmannii CBS 245.38. Genome Announc. 6
Leonhardt, S., Büttner, E., Gebauer, A.M., Hofrichter, M., Kellner, H.
(Siehe online unter https://doi.org/10.1128/genomeA.01510-17) - 2019. Genome and secretome of Chondrostereum purpureum correspond to saprotrophic and phytopathogenic life styles. PLOS ONE 14, e0212769
Reina, R., Kellner, H., Hess, J., Jehmlich, N., García-Romera, I., Aranda, E., Hofrichter, M., Liers, C.
(Siehe online unter https://doi.org/10.1371/journal.pone.0212769) - 2020. Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions, in: Nevalainen, H. (Ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology. Springer International Publishing, Cham, pp. 369–403
Hofrichter, M., Kellner, H., Herzog, R., Karich, A., Liers, C., Scheibner, K., Kimani, V.W., Ullrich, R.
(Siehe online unter https://doi.org/10.1007/978-3-030-29541-7_14) - 2020. Unspezifische Peroxygenasen — Oxyfunktionalisierung außerhalb der Pilzhyphe. Biospektrum 26, 103–106
Scheibner, K., Ullrich, R., Kiebist, J., Kellner, H., Hofrichter, M.
(Siehe online unter https://doi.org/10.1007/s12268-020-1338-x)
