Fig. 2

Xylanolytic activities of yeast strains engineered with CRISPR/Cas9. (A) Schematic map of the plasmid pJR2_04_SED1-XylA-BmXyn11A-Agu115 containing XylA β-xylosidase, BmXyn11A xylanase and Agu115 α-methyl-glucuronidase genes with NotI plasmid linearization sites adjacent to homology arms for homologous recombination into the S. cerevisiae genome at the X2 locus. (B) Secreted xylanase activity from cell-free supernatants of yeast strains grown in 2 mL YPD and incubated in 10 g L^− 1 beechwood GX compared to purified recombinant 1 mg mL^− 1BmXyn11A using DNS reducing sugar assays in triplicates. Strain names indicate what recombinant enzyme is engineered e.g. xylanase (BmXyn11A, XynHB or XynB). (C) Clearing zones (indicated by red arrows) on agar plates containing Delft medium with 8 g L^− 1 beechwood GX mediated by heterologous xylanase secretion from co-expression strains after 48 h incubation at 30 °C using a 10 µL drop with OD = 5 cell density. (D) Subcellular β-xylosidase activity quantified using p-nitrophenyl-β-D-xylopyranoside and (E) subcellular α-methyl-glucuronidase activity determined by NADH-based D-glucuronic acid in duplicates. Values are means ± standard deviations as error bars. Asterisks indicate statistical significance in subcellular activity levels between the XXX strain and engineered strains. P values ≤ 0.05 (*), ≤ 0.01 (**) and ≤ 0.001 (***) were considered significant (n = 2–3) and evaluated using one-way ANOVA Dunnet’s test with XXX fractions as control group