Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): a key to cellulase gene expression on lactose

Abstract

The heterodisaccharide lactose (1,4-O-beta-D-galactopyranosyl-D-glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei). Lactose assimilation is slow, and the assimilation of its beta-D-galactose moiety depends mainly on the operation of a recently described reductive pathway and depends less on the Leloir pathway, which accepts only alpha-D-galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular beta-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the beta-anomer of D-galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.

Fig. 1.

Transcription profile of aep1, aep2, and aep3 of H. jecorina QM9414 (QM) during growth on different carbon sources obtained by RT-PCR. Glc, glucose; Gal, galactose; Lac, lactose; M, marker.

Fig. 2.

Demonstration of the presence and expression of GAL10^1068-2100 in H. jecorinh mutants. (A) Schematic drawing of the GAL10^1068–2100 expression cassette used for the transformation of H. jecorina TU-6. (B) Demonstration of the presence of S. cerevisiae GAL10^1068–2100 in the H. jecorina mutant strains M1, M2a, and M2b by PCR and its absence in QM9414. M, marker; Cont., control (pGAL10_Kicsi used as a template). (C) Transcription of the S. cerevisae GAL10^1068–2100 in H. jecorina strains M1, M2a, and M2b during growth on lactose at 5 and 27 h. Y, S. cerevisiae (positive control); QM, H. jecorina QM9414; M, marker; Lac, lactose. (D) Specific intracellular mutarotase activity of the QM9414- and GAL10^1068–2100-expressing H. jecorina strains. Samples were taken 12 h after the transferring procedure.

Fig. 3.

Fermentation profiles of H. jecorina QM9414 and mutants M1, M2a, and M2b on different carbon sources. (A) Fermentation profile of H. jecorina QM9414 (circles) and the GAL10^1068–2100-expressing strains M1 (squares), M2a (triangles), and M2b (diamonds) on glucose as a sole carbon source. Open symbols indicate glucose, and filled symbols indicate biomass time profiles. (B) Fermentation profile of H. jecorina QM9414 (circles) and strains M1 (squares) M2a (triangles), and M2b (diamonds) on galactose as a sole carbon source. Open symbols indicate galactose, and filled symbols indicate biomass time profiles. (C) Lactose uptake of H. jecorina QM9414 (○) and the GAL10^1068–2100-expressing strains M1 (□) M2a (▵), and M2b (◇). (D) Biomass formation of H. jecorina QM9414 (●) and the GAL10^1068–2100-expressing strains M1 (■), M2a (▴), and M2b (♦) on lactose.

Fig. 4.

Specific extracellular β-galactosidase activity of H. jecorina QM9414 and GAL10^1068–2100-expressing strains M1, M2a, and M2b. Samples were taken 12 h after the transferring procedure.

Fig. 5.

Expression of the H. jecorina genes gal1 (encoding a galactokinase) and xyl1 (encoding an aldose reductase) on lactose in the QM9414 and GAL10^1068–2100-expressing H. jecorina strains M1, M2a, and M2b.

Fig. 6.

Expression of H. jecorina cbh1 and cbh2 genes on different carbon sources. (A) Expression of the H. jecorina cellulase genes cbh1 and cbh2 on lactose at 27 h and at 40 h in the QM9414 and GAL10^1068–2100-expressing H. jecorina strains M1, M2a, and M2b. (B) Expression of the H. jecorina cellulase genes cbh1 and cbh2 on cellulose at 40 h in the QM9414 and GAL10^1068–2100-expressing H. jecorina strains M1, M2a, and M2b.