D-Xylose as a repressor or inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei)

Abstract

For Hypocrea jecorina (anamorph Trichoderma reesei), a filamentous fungus used for hydrolase production in different industries, it has been a long-term practice to use d-xylose as an inducing substance. We demonstrate in this study that the degree of xylanase-encoding gene induction strictly depends on the concentration of d-xylose, which was found to be optimal from 0.5 to 1 mM for 3 h of cultivation. At higher concentrations of d-xylose, a reduced level of xylanase gene expression was observed. In the present study, we also provide evidence that the d-xylose concentration-dependent induction is antagonized by carbon catabolite repressor 1. This repressor mediates its influence on d-xylose indirectly, by reducing the expression of xylanase regulator 1, the main activator of most hydrolase-encoding genes. Additionally, a direct influence of the repressor on xylanase 1 expression in the presence of d-xylose was found. Furthermore, we show that d-xylose reductase 1 is needed to metabolize d-xylose to achieve full induction of xylanase expression. Finally, a strain which expresses xylanase regulator 1 at a constant level was used to partially overcome the negative influence exerted by carbon catabolite repressor 1 on d-xylose.

FIG. 1.

Analysis of d-xylose concentration-dependent transcription of xylanase-encoding genes in H. jecorina. The parental strain QM9414 was precultured on glycerol and was thereafter transferred to MA medium without a carbon source (NC) or containing 1% (wt/vol) glycerol (GY) or the indicated d-xylose concentration as the sole carbon source and was incubated for 3 h. Transcription analyses of xyn1 (A), xyn2 (B), and bxl1 (C) were performed via qPCR, using actin transcription for normalization. Results are given as relative transcript ratios on a logarithmic scale (lg). The values provided in the figures are means from three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment, which is indicated by an asterisk.

FIG. 2.

Transcription analysis of d-xylose-dependent Cre1 influence on xylanase-encoding genes in H. jecorina. The parental strain QM9414 (dark gray bars) and a Δcre1 strain (light gray bars) were precultured on glycerol and were thereafter transferred to MA medium containing 1% (wt/vol) glucose (G) or the indicated d-xylose concentration as the sole carbon source, mixtures of 1% (wt/vol) glucose with the indicated d-xylose concentrations, or no carbon source (NC) and incubated for 3 h. Transcription analyses of xyn1 (A), xyn2 (B), and bxl1 (C) were performed via qPCR, using actin transcription for normalization. Results are given as relative transcript ratios on a logarithmic scale (lg). The values given in the figures are means for three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment, which is indicated by an asterisk. ND, no transcript detected.

FIG. 3.

Analysis of Cre1-dependent d-xylose influence on xyr1 transcription in H. jecorina. The parental strain QM9414 (dark gray bars) and a Δcre1 strain (light gray bars) were precultured on glycerol and were thereafter transferred to MA medium containing 1% (wt/vol) glucose (G) or the indicated d-xylose concentration as the sole carbon source or no carbon source (NC) and then incubated for 3 h. Remaining concentrations of d-xylose after 3 h of incubation are given in parentheses (QM9414/Δcre1 strain) below the starting concentration. Transcription analysis of xyr1 was performed via qPCR, using actin transcription for normalization. Results are given as relative transcript ratios on a logarithmic scale (lg). The values given in the figures are means for three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment, which is indicated by an asterisk.

FIG. 4.

Transcription analysis of optimal d-xylose induction of xylanase-encoding genes in a Cre1-negative background of H. jecorina. The parental strain QM9414 and a Δcre1 strain were precultured on glycerol and were thereafter transferred to MA medium containing the indicated d-xylose concentration as the sole carbon source and then incubated for 3 h. Transcription analyses of xyn1, xyn2, and bxl1 were performed via qPCR, using actin transcription for normalization. Results are given as the logarithmic transcript ratio for the Δcre1 strain to QM9414. The values given in the figures are means for three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment (QM9414, no carbon source, 3 h).

FIG. 5.

Transcription analysis of optimal d-xylose induction of xylanase-encoding genes in a Xyl1-negative background of H. jecorina. The parental strain QM9414 and a Δxyl1 strain were precultured on glycerol and were thereafter transferred to MA medium containing the indicated d-xylose concentration as the sole carbon source and then incubated for 3 h. Transcription analyses of xyn2 and bxl1 were performed via qPCR, using actin transcription for normalization. Results are given as the logarithmic transcript ratio for the Δxyl1 strain to QM9414. The values given in the figures are means for three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment (QM9414, no carbon source, 3 h).

FIG. 6.

Transcription analysis of xylanase-encoding genes in H. jecorina at high d-xylose concentrations when xyr1 was constitutively expressed. The parental strain QM9414 and the nx7 strain were precultured on glycerol and were thereafter transferred to MA medium containing 66 mM d-xylose as the sole carbon source and then incubated for 3 h. Transcription analyses of xyn1, xyn2, and bxl1 were performed via qPCR, using actin transcription for normalization. Results are given as the logarithmic transcript ratio for the nx7 strain to QM9414. The values given in the figures are means for three independent experiments. Error bars indicate standard deviations. Transcript levels always refer to one reference sample within an experiment (QM9414, no carbon source, 3 h).

FIG. 7.

Schematic drawing of interrelated mechanisms underlying the effects of d-xylose on xylanase expression in H. jecorina. +, positive regulation; −, negative regulation.