Background: Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes.Aspergillus nidulans,a well-studied saprophyte and close homologue to industrially important species such asA. nigerandA. oryzae, was selected for this study.Results: A. nidulanswas grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content,A. nidulansgrew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome ofA. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.Conclusions: Our results show thatA. nidulanssecretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulansis capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.

Myceliophthora thermophyla is a thermophilic industrially relevant fungus that secretes an assortment of hydrolytic and oxidative enzymes for lignocellulose degradation. Among them is glyoxal oxidase (MtGLOx), an extracellular oxidoreductase that oxidizes several aldehydes and α-hydroxy carbonyl substrates coupled to the reduction of O₂ to H₂O₂. This copper metalloprotein belongs to a class of enzymes called radical copper oxidases (CRO) and to the “auxiliary activities” subfamily AA5_1 that is based on the Carbohydrate-Active enZYmes (CAZy) database. Only a few members of this family have been characterized to date. Here, we report the recombinant production, characterization, and structure-function analysis of MtGLOx. Electron Paramagnetic Resonance (EPR) spectroscopy confirmed MtGLOx to be a radical-coupled copper complex and small angle X-ray scattering (SAXS) revealed an extended spatial arrangement of the catalytic and four N-terminal WSC domains. Furthermore, we demonstrate that methylglyoxal and 5-hydroxymethylfurfural (HMF), a fermentation inhibitor, are substrates for the enzyme.

Lytic polysaccharide monooxygenases (LPMOs) are copper dependent enzymes that carry out oxidative cleavage of cellulose and other polysaccharides. Aspergillus nidulans, an ascomycete fungus that contains multiple AA9 LPMOs in the genome, offers an excellent model system to study their activity during the oxidative degradation of biomass. AN1602, a dual domain AA9-LPMO in A. nidulans appended with a carbohydrate-binding module, CBM1, was expressed in Pichia pastoris for analyzing oxidative cleavage on cellulosic substrates. The mass spectral and HPAEC analyses showed that the enzyme cleaves phosphoric acid swollen cellulose (PASC) in the presence of a reducing agent, yielding a range of cello-oligosaccharides. In addition to the polymeric substrate cellulose, AN1602 is also active on soluble cellohexaose, a property that is restricted to only a few characterized LPMOs. Product analysis of AN1602 cleaved cellohexaose revealed that C4 was the sole site of oxidation. The sequence and predicted structure of the catalytic domain of AN1602 matched very closely to known C4 cellohexaose active enzymes.