Trichoderma reesei has been of industrial interest for several decades for the production of cellulases, resulting in the development of hypercellulolytic strains such as RUT-C30. High protein production and secretion, and eukaryotic glycosylation machinery, make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients and this variability in glycosylation affects stability and activity of CBHI, the main secreted protein. However, the impact of media on the intracellular precursors of glycosylation and on the secreted protein glycosylation has received little attention, and techniques for their analysis in fungi are lacking.

 This study investigated the effect of carbon source on nucleotide sugars of RUT-C30, important precursors of glycosylation. Methods were developed to extract and analyse these metabolites from mycelia grown on glucose or lactose. In parallel, the glycosylation profile of secreted protein was monitored. Differences were found in the abundance of nucleotide sugars depending on carbon source, not directly reflected in secreted protein glycosylation. In addition, previously unidentified O-glycan structures on the total secreted protein were identified. The work provides insight into the effect of carbon sources on the glycosylation and protein secretion pathways in Trichoderma reesei RUT-C30.