L-arabitol is the actual inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei)

Abstract

The saprophytic fungus Hypocrea jecorina (anamorph, Trichoderma reesei) is an important native producer of hydrolytic enzymes, including xylanases. Regarding principles of sustainability, cheap and renewable raw materials, such as d-xylose (the backbone monomer of xylan), have been receiving increasing attention from industries. Recently, it was demonstrated that small (0.5 to 1 mM) amounts of d-xylose induce the highest expression of xylanase in H. jecorina. However, it was also reported that active metabolism of d-xylose is necessary for induction. In this report, we demonstrate that xylitol, the next intermediate in the pentose pathway after d-xylose, does not trigger transcription of xylanase-encoding genes in H. jecorina QM9414. The highest level of transcription of xylanolytic enzyme-encoding genes occurred in an xdh1 (encoding a xylitol dehydrogenase) deletion strain cultured in the presence of 0.5 mM d-xylose, suggesting that a metabolite upstream of xylitol is the inducer. The expression levels of xylanases in an xdh1-lad1 double-deletion strain were lower than that of an xdh1 deletion strain. This observation suggested that l-xylulose is not an inducer and led to the hypothesis that l-arabitol is the actual inducer of xylanase expression. A direct comparison of transcript levels following the incubation of the H. jecorina parental strain with various metabolites of the pentose pathway confirmed this hypothesis. In addition, we demonstrate that xyr1, the activator gene, is not induced in the presence of pentose sugars and polyols, regardless of the concentration used; instead, we observed low constitutive expression of xyr1.

Fig. 1.

Schematic drawing of the pentose pathway in H. jecorina. Metabolites are shown in boxes. The names of genes and EC numbers of their corresponding enzymes are indicated. Solid lines indicate one-step reactions; dashed lines indicate multiple-step reactions.

Fig. 2.

Comparative transcript ratio analysis of Xyl1-dependent influence on xylanase-encoding genes in H. jecorina. (A) Overview of the pathways blocked by deleting xyl1. AO, l-arabinose; AL, l-arabitol; L-XY, l-xylulose; XO, d-xylose; XL, xylitol; D-XY, d-xylulose; D-XY-5P, d-xylulose-5-phosphate. The parental strain QM9414 and a Δxyl1 strain were precultured with glycerol and were later transferred to MA medium without a carbon source (B) or to MA medium containing 0.5 mM (C), 5 mM (D), 10 mM (E), or 66 mM (F) xylitol as the sole carbon source. Transcript ratios for the xyn1, xyn2, bxl1 and xyr1 genes were calculated using REST 2009 software. The dashed line (ratio of 1) indicates equal amounts of transcript in the two strains. Values above 1 indicate higher transcription in the Δxyl1 strain compared to QM9414, and values below 1 indicate lower transcription. Error bars represent 95% confidence intervals.

Fig. 3.

Comparative transcript ratio analysis of Xdh1-dependent influence on xylanase-encoding genes in H. jecorina. (A) Overview of the pathways blocked by deleting xdh1. AO, l-arabinose; AL, l-arabitol; L-XY, l-xylulose; XO, d-xylose; XL, xylitol; D-XY, d-xylulose; D-XY-5P, d-xylulose-5-phosphate. The parental strain QM9414 and a Δxdh1 strain were precultured with glycerol and were later transferred to MA medium without a carbon source (B) or to MA medium containing 0.5 mM (C), 5 mM (D), 10 mM (E), or 66 mM (F) d-xylose as the sole carbon source. Transcript ratios for the xyn1, xyn2, bxl1, and xyr1 genes were calculated using REST 2009 software. The dashed line (ratio of 1) indicates equal amounts of transcript in the two strains. Values above 1 indicate higher transcription in the Δxdh1 strain compared to QM9414, and values below 1 indicate lower transcription. Error bars indicate 95% confidence intervals.

Fig. 4.

Comparative transcript ratio analysis of Xdh1- and Lad1-dependent influence on xylanase-encoding genes in H. jecorina. (A) Overview of the pathways blocked by deleting xdh1 and lad1. AO, l-arabinose; AL, l-arabitol; L-XY, l-xylulose; XO, d-xylose; XL, xylitol; D-XY, d-xylulose; D-XY-5P, d-xylulose-5-phosphate. The Δxdh1 strain and the Δxdh1-lad1 strain were precultured on glycerol and were later transferred to MA medium without a carbon source (B) or to MA medium containing 0.5 mM (C), 5 mM (D), 10 mM (E), or 66 mM (F) d-xylose as the sole carbon source. Transcript ratios for the xyn1, xyn2, bxl1, and xyr1 genes were calculated using REST 2009 software. The dashed line (ratio of 1) indicates equal amounts of transcript in the two strains. Values above 1 indicate higher transcription in the Δxdh1 strain compared to the Δxdh1-lad1 strain, and values below 1 indicate lower transcription. Error bars represent 95% confidence intervals.

Fig. 5.

Comparative transcript ratio analysis of xylanase-encoding genes in H. jecorina by the use of different pentoses. The QM9414 parental strain was precultured on glycerol and was thereafter transferred to and incubated in MA medium without a carbon source (A) or in MA medium containing 0.5 mM l-arabinose (B), d-xylulose (C), xylitol (D), l-xylulose (E), or l-arabitol (F) as the sole carbon source. Transcript ratios for the xyn1, xyn2, bxl1, and xyr1 genes were calculated using REST 2009 software. ND, not detected. The dashed line (ratio of 1) indicates equal amounts of transcript in the two strains. Values above 1 indicate higher transcription on the corresponding pentose compared to d-xylose, and values below 1 indicate lower transcription. Error bars represent 95% confidence intervals.