Avaliação e padronização da técnica de espectrometria de massa do tipo MALDI-TOF para identificação de Basidiomicetos (Hymenochaetaceae e Polyporaceae) e prospecção de peptídeos bioativos

Abstract

Fungi comprise a group of organisms that have high taxonomic and functional diversity, and therefore, are of great biotechnological interest. They can act in wood decomposition and industrial waste, playing a fundamental role in the nutrients cycling. In addition, they produce various biomolecules derived from primary and secondary metabolism, such as polysaccharides, peptides, enzymes, among others, which perform, for example, antitumor, anti-inflammatory, immunomodulatory and antimicrobial activity. Estimates suggest that there are 5.1 million fungal species in the world, of which approximately 10% have been described so far. Hymenochaetaceae and Polyporaceae, which are part of the phylum Basidiomycota, can act as plant pathogens, produce enzymes of industrial interest, and playan important role in the degradation of organic matter. Despite the great diversity and economic importance of fungi, this is still a group of microorganisms that needs to be further studied. Thus, studies related to the taxonomic identification of species, as well as those focused on the prospection and identification of bioactive molecules produced by these organisms are of great interest and importance, both from a scientific and commercial point of view. Currently, the identification of fungi at the species level is based on morphological (macroscopic and microscopic) and molecular identification by DNA barcoding (gold standard). The first is dependent on specialized professionals (mycologists) and the second is expensive, time consuming and susceptible to environmental contamination. In the last decades, mass spectrometry has emerged as an alternative for the identification of bacteria and fungi of clinical interest, however, few studies have used this technique in the identification of fungi of biotechnological interest. In this context, the goals of this work were (i) to standardize the taxonomic identification of fungi of the families Hymenochaetaceae and Polyporaceae using the mass spectrometry tool (MALDI-TOF MS), (ii) to create a spectra reference database (protein fingerprints) for the identification of species of these two families,and (iii) to prospect and identify peptides with biotechnological potential. Herein, we demonstrate that the best protocol for obtaining the mass spectrum is one that eliminates the step of pre-extracting proteins and uses the mycelium directly on the MALDI target plate (intact cell mass spectrometry). An in-house database was created using 28 isolates previously identified in this work using morphological characters (classical taxonomy) and the ITS region of rDNA. After this database had built, it was tested using Bruker's real-time identification tool, and all the isolates tested had a score > 2.000, indicating a reliable identification at the species level. We conclude that this technique is highly efficient for the identification of the two families tested at the species level and is sufficiently robust to differentiate closely related taxa. Extracts containing the metabolites of the isolates Trametes villosa (CCMB561), Ganoderma sp. 1 (DHCR379) and Fulvifomes nilgheriensis (GAS917) were produced to prospect peptaibols, however, none of the three isolates demonstrated potential for the production of these peptides under the conditions tested.