Supplementing XYR1-mutated Trichoderma reesei strain cultivation with (SO2-ethanol-water) softwood pulp improves cellulase production

The cellulolytic enzyme cost remains a major bottleneck in converting lignocellulose, especially softwoods, into fuels and chemicals. The aim of this study was to evaluate possibilities to increase enzyme production efficiency by using SO ₂-ethanol-water (SEW) pretreated softwood pulp with a Trichoderma reesei strain that expresses a mutant form of the main transcriptional regulator, XYR1, of cellulase- and hemicellulase genes leading to loss of glucose repression/carbon catabolite repression. The (hemi)cellulase enzyme cocktail of this strain was improved by expressing three heterologous enzymes, a beta-glucosidase, a CEL6 (CBH2) exoglucanase and a lytic polysaccharide mono‑oxygenase. Seven bioreactor cultivations were performed using glucose and different cellulose supplementations and glucose feed strategies. We showed that adding 3 %-w/v cellulose to the glucose medium and starting the glucose feed when the glucose was consumed from the batch medium, improved the protein production rate by over 80 % during the first five days compared to total absence of cellulose. With only 3 % cellulose addition to the batch phase, we estimate that over one third of time and total carbon source, including cellulose, could be saved compared to a production process without cellulosic substrate supplementation. Additionally, enzymes produced with SEW pulp in 119 h and those produced with glucose alone in 193 h both achieved 90 % glucose conversion when used for SEW pulp hydrolysis at a protein loading of 4–5 mg/g cellulose. Herein, we have shown that the M2883 strain can produce more than 29 FPU/mL of the complete set of cellulase enzymes both with and without cellulose supplementation.