Accessory enzymes of hypercellulolytic Penicillium funiculosum facilitate complete saccharification of sugarcane bagasse

Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig1.sup.88, in response to SCB induction and examined their role in the saccharification of SCB. A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0-45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 280 proteins were identified in the secretome of PfMig1.sup.88, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-[beta]-1,3(4)-glucanase (GH16), endo-[alpha]-1,3-glucanase (GH71), xylanase (GH30), [beta]-xylosidase (GH5), [beta]-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in [beta]-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB. Our findings provide an insight into the enzyme system of PfMig1.sup.88 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.