A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation

Abstract

Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

Keywords: (Hemi)cellulases; Biopolymer degradation; Hydrolases; Hypocrea jecorina; Trichoderma reesei; Xyr1.

Fig. 1

Growth and clearing properties of H. jecorina QM9414 and nx7. (A) Growth rates of both strains have been determined by cultivation on agar plates containing the indicated carbon source or biopolymer at 30 °C. Additionally, the rate of clearing of the xylan-containing medium was determined by measuring the clearing zone. Values are means of three biological replicates; standard deviation was below 5%. (B) Pictures of H. jecorina QM9414 and nx7 strains on the plates used for determination of growth rates.

Fig. 2

Analysis of the conversion of biopolymers to mono- and disaccharides via enzymatic catalysis using supernatants from H. jecorina fermentations on birch wood xylan. The biopolymers birch wood xylan (A and D), beech wood xylan (B and E), and M. giganteus (C and F) were incubated with supernatants from H. jecorina nx7 (light grey) and QM9414 (dark grey) for 0, 20, 40, 60, 120, 180 min at 40 °C. Afterwards HPLC-analysis was performed using d-xylose (A–C) and xylobiose (D–F) as standards. Values are means of four replicates (two biological replicates used in duplicates in the enzyme assay); standard deviation was below 5%.

Fig. 3

Analysis of the conversion of biopolymers to mono- and disaccharides via enzymatic catalysis using supernatants from H. jecorina fermentations on CMC. The biopolymers birch wood xylan (A and D), beech wood xylan (B and E), and M. giganteus (C and F) were incubated with supernatants from H. jecorina nx7 (light grey) and QM9414 (dark grey) for 0, 20, 40, 60, 120, 180 min at 40 °C. Afterwards HPLC-analysis was performed using d-xylose (A–C) and xylobiose (D–F) as standards. Values are means of four replicates (two biological replicates used in duplicates in the enzyme assay); standard deviation was below 5%.

Fig. 4

Analysis of the conversion of crystalline cellulose to its monosaccharide via enzymatic catalysis using supernatants from different H. jecorina fermentations. The biopolymer was incubated with supernatants from H. jecorina nx7 (light grey) and QM9414 (dark grey) fermentations on birch wood xylan (A) and CMC (B) for 0, 20, 40, 60, 120, 180 min at 40 °C. Afterwards HPLC-analysis was performed using glucose as standard. Values are means of four replicates (two biological replicates used in duplicates in the enzyme assay); standard deviation was below 5%.

Fig. 5

Analysis of the conversion of cellulosic biopolymers to its monosaccharide via enzymatic catalysis using supernatants from H. jecorina fermentations on birch wood xylan. The biopolymers CMC (A) and crystalline cellulose (B) were incubated with supernatants from H. jecorina nx7 (light grey) and QM9414 (dark grey) fermentations for 0, 20, 40, 60, 120, 180 min at 40 °C. Afterwards HPLC-analysis was performed using glucose as standard. Values are means of four replicates (two biological replicates used in duplicates in the enzyme assay); standard deviation was below 5%.