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Background: Fermentation of sugars derived from plant biomass feedstock is crucial for sustainability. Hence, utilizing customized enzymatic cocktails to obtain oligosaccharides instead of monomers is an alternative fermentation strategy to produce prebiotics, cosmetics, and biofuels. This study developed an engineered strain of Aspergillus niger producing a tailored cellulolytic cocktail capable of partially degrading sugarcane straw to yield cellooligosaccharides., Results: The A. niger prtT∆ strain created resulted in a reduced extracellular protease production. The prtT∆ background was then used to create strains by deleting exoenzyme encoding genes involved in mono- or disaccharide formation. Consequently, we successfully generated a tailored prtT∆bglA∆ strain by eliminating a beta-glucosidase (bglA) gene and subsequently deleted two cellobiohydrolases and one beta-xylosidase encoding genes using a multiplex strategy, resulting in the Quintuple∆ strain (prtT∆; bglA∆; cbhA∆; cbhB∆; xlnD∆). When applied for sugarcane biomass degradation, the tailored secretomes produced by A. niger resulted in a higher ratio of cellobiose and cellotriose compared with glucose relative to the reference strain. Mass spectrometry revealed that the Quintuple∆ strain secreted alternative cellobiohydrolases and beta-glucosidases to compensate for the absence of major cellulases. Enzymes targeting minor polysaccharides in plant biomass were also upregulated in this tailored strain., Conclusion: Tailored secretome use increased COS/glucose ratio during sugarcane biomass degradation showing that deleting some enzymatic components is an effective approach for producing customized enzymatic cocktails. Our findings highlight the plasticity of fungal genomes as enzymes that target minor components of plant cell walls, and alternative cellulases were produced by the mutant strain. Despite deletion of important secretome components, fungal growth was maintained in plant biomass., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications., Results: In this study, BxlB-a highly secreted GH3 β-xylosidase from A. nidulans , presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N -glycans on BxlB secretion and function. The non-glycosylated mutant (BxlB non-glyc ) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB wt ), while a partially glycosylated mutant (BxlB N1;5;7 ) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlB CC ), despite the high transcript levels. BxlB wt , BxlB non-glyc , and BxlB N1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlB N1;5;7 , to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB N1;5 and BxlB N5;7 ) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlB non-glyc and BxlB N1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlB non-glyc and BxlB N1;5 ., Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)