Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification.

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Analysis of Trichophyton rubrum gene expression in response to cytotoxic drugs.

Suppressive subtractive hybridization was used to isolate transcripts specifically upregulated during Trichophyton rubrum exposure to acriflavin, fluconazole, griseofulvin, terbinafine or undecanoic acid. Macro-array dot-blot and sequencing of 132 clones, which correspond to genes differentially expressed after exposition of T. rubrum to at least one of these cytotoxic drugs, revealed 39 unique genes. Of these, 32 have not been previously described in T. rubrum, representing an increase in the number of T. rubrum genes that have been identified. The upregulation of the novel genes encoding a retrotransposon element, a carboxylic ester hydrolase, a copper resistance-associated P-type ATPase, a DNA mismatch repair protein and a NIMA (never in mitosis A) interactive protein was confirmed by Northern blot.