Phylogenetic and functional diversity of fungal heme peroxidases – PEROXIDIV

Analysis of peroxidase diversity necessitates the implementation of bioinformatics and phylogenetic approaches to (1) identify the corresponding genes within public databases and (2) infer the evolutionary history of these protein families. Confrontation between “real” and “known” diversity could then be used to identify sequences on which to concentrate to carry out further functional characterizations.
The characterization of peroxidase genes expressed in environmental samples necessitated the implementation of RNA extraction protocols from decaying wood, soil and sediments. Oligonucleotidic probes representative of the studied gene families were designed in order to capture by hybridization the environmental sequenced that were sequenced using high-throughput technologies.
The functional characterization of new peroxidases, including those from the environment, necessitates their production as active recombinant proteins in a microbial host. Production of this class of enzymes is often problematic and different bacterial and fungal hosts (including yeasts and filamentous fungi) had been tested.

Fungal DyP and UPO protein families display an unsuspected 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 bioinformatics tools from high-throughput 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). Only this las host allowed production of one enzyme in an active form.

The results obtained underline the complementarity of the different approaches, that encompassed data mining, molecular phylogeny and environmental genomics to highlight the global structural diversity of understudied protein families present a strong potential in biotechnology. The production of the corresponding enzymes in heterologous hosts for their functional characterization still remains a challenge to overcome

Fungal heme peroxidases and peroxygenases are found in 3 distinct protein families. This large genetic pool represents a unique source of functional catalytic diversity, which has already being exploited in industry but needs to be further explored to foster its broader application. PeroxiDiv associates 3 partners with competences in environmental genomics, bioinformatics, biochemistry/enzymology and biotechnology to reveal, by an original approach of targeted sequence capture, the diversity of peroxidase-encoding genes expressed by fungal communities in distinct environments (grassland and forest soils, wood). This approach will allow the isolation of a large number of so-far unknown peroxidases with possibly novel biochemical properties. Their analysis in silico (phylogeny, 3D modeling) will lead to the identification of new variants that will be overexpressed and characterized with respect to their catalytic performance (substrate spectra, reaction products, stability, etc.).