Bioprospecção e biotransformação de substâncias bioativas: explorando o potencial biotecnológico de fungos filamentosos

Abstract

Obtaining biologically active compounds from fungal metabolism has been widely reported, but the number of fungal species studied remains below the estimated number. Fungi of the genus Botrytis are widely known for their efficient ability to produce diseases in plants of agricultural and commercial interest. Botrytis species express similar behaviors towards the plants they infect, however, B. deweyae presents unique characteristics within the genus, being able to behave as an endophyte. Specialized metabolites such as botrydial (a substance with phytotoxic properties) or its derivatives have already been isolated from B. cinerea, but no metabolite has yet been characterized from B. deweyae cultures. For these reasons, the first chapter of the present work consisted of the first study on the specialized metabolism of this species. The analysis of the fermentative extract resulted in the isolation and characterization of seven substances derived from botrydial. The interest in filamentous fungi is not restricted to the study of specialized metabolism. The potential for the production and secretion of diverse enzymes places such microorganisms as unique catalytic agents that lead to selective chemical modifications. In biotransformation, fungi are used as catalysts for unusual reactions, which would be difficult to obtain and generally lead to regio-, chemo- and stereoselectivity, using ecologically correct conditions. As a way of studying the biocatalytic potential of different species of filamentous fungi, the second chapter of this work selected two model substrates to map the main reactions catalyzed by the pre-selected strains. The substrates used were progesterone, a steroid with a poorly functionalized carbon chain, and hydroxychloroquine, a drug with anti-inflammatory and antiviral activities, which was part of certain clinical trials against Sars-CoV-2 during the COVID-19 pandemic. Endophytic fungi isolated from the leaves of Handroanthus impetiginosus (purple ipê) were used in the biotransformation of progesterone and five derivatives were noted from the analysis of their mass data. The endophyte Talaromyces sp. H4 was able to transform the steroid nucleus of progesterone selectively into four products through selective reduction of the C4-C5 double bond and oxidation of the C-17 position. The best progesterone conversion rate (>90%) was achieved with the endophytic strain Penicillium citrinum H7, which biotransformed the substrate exclusively into a single hydroxylated product. Finally, the biotransformation of hydroxychloroquine was studied using filamentous fungi reported as microbial models to study the in vitro metabolism of drugs, capable of mimicking mammalian metabolism and providing metabolites used in the assessment of cytotoxicity. Cunninghamella echinulata var. elegans ATCC 8688a metabolized hydroxychloroquine into a main metabolite identified as 4-(1,2,3,4- tetrahydroquinoline-4-ylamino)pentan-1-ol, a new substance in the literature. The microbial transformation catalyzed N-dealkylation, 7-chloro-elimination and reduction of the two conjugated double bonds of the quinoline system of hydroxychloroquine. The cytotoxic profiles of hydroxychloroquine and its metabolite were evaluated using CCD-1059Sk cells (human fibroblasts), showing that both substances exhibited cytotoxic activities in CCD-1059Sk, but the metabolite obtained after biotransformation was significantly more toxic. In general, the results shown here highlight the efficiency and versatility of fungal applications in obtaining substances of interest in different areas, mainly in bioprospecting and biotransformation.