Molecular regulation of arabinan and L-arabinose metabolism in Hypocrea jecorina (Trichoderma reesei)

Abstract

Hypocrea jecorina (anamorph: Trichoderma reesei) can grow on plant arabinans by the aid of secreted arabinan-degrading enzymes. This growth on arabinan and its degradation product L-arabinose requires the operation of the aldose reductase XYL1 and the L-arabinitol dehydrogenase LAD1. Growth on arabinan and L-arabinose is also severely affected in a strain deficient in the general cellulase and hemicellulase regulator XYR1, but this impairment can be overcome by constitutive expression of the xyl1 encoding the aldose reductase. An inspection of the genome of H. jecorina reveals four genes capable of degrading arabinan, i.e., the alpha-L-arabinofuranosidase encoding genes abf1, abf2, and abf3 and also bxl1, which encodes a beta-xylosidase with a separate alpha-L-arabinofuranosidase domain and activity but no endo-arabinanase. Transcriptional analysis reveals that in the parent strain QM9414 the expression of all of these genes is induced by L-arabinose and to a lesser extent by L-arabinitol and absent on D-glucose. Induction by L-arabinitol, however, is strongly enhanced in a Deltalad1 strain lacking L-arabinitol dehydrogenase activity and severely impaired in an aldose reductase (Deltaxyl1) strain, suggesting a cross talk between L-arabinitol and the aldose reductase XYL1 in an alpha-L-arabinofuranosidase gene expression. Strains bearing a knockout in the cellulase regulator xyr1 do not show any induction of abf2 and bxl1, and this phenotype cannot be reverted by constitutive expression of xyl1. The loss of function of xyr1 has also a slight effect on the expression of abf1 and abf3. We conclude that the expression of the four alpha-L-arabinofuranosidases of H. jecorina for growth on arabinan requires an early pathway intermediate (L-arabinitol or L-arabinose), the first enzyme of the pathway XYL1, and in the case of abf2 and bxl1 also the function of the cellulase regulator XYR1.

FIG. 1.

l-Arabinose catabolic pathway of fungi. The question mark indicates a step for which the respective gene has not been found yet (see, for example, the study by Metz et al. [28]).

FIG. 2.

Radial growth of H. jecorina on various carbon sources on plates (a) and biomass production in submerged cultures (b). Carbon sources are indicated as follows: d-glucose (○), l-arabinose (▴), l-arabinitol (⋄), and arabinan (⧫), all at 1% (wt/vol).

FIG. 3.

Growth of H. jecorina QM9414 and strains deleted in different steps of the l-arabinose catabolic pathway on d-glucose (a), l-arabinose (b), l-arabinitol (c), and arabinan (d). Strains are indicated as follows: H. jecorina QM9414 (⧫), Δxyl1 (▪), and Δlad1 (▴).

FIG. 4.

Effect of the loss of XYR1 on growth of H. jecorina on l-arabinose and l-arabinitol. (a) Growth of H. jecorina QM9414 (solid symbols) and Δxyr1 (open symbols) on l-arabinose (triangles), l-arabinitol (squares), and arabinan (diamonds) as the carbon source. (b) Growth of H. jecorina QM9414 (▴, ⧫) and Ptef1:xyl1/Δxyr1 (○, ×) strains on l-arabinose and arabinan, respectively. (c) Growth of H. jecorina QM9414, Δxyr1, and Ptef1:xyl1/Δxyr1 strains on plates containing l-arabinose as the sole carbon source.

FIG. 5.

Phylogenetic analysis of H. jecorina ABF1 and ABF3. The other sequences used were identified as those showing highest similarity in BLAST. Neighbor joining was used, and gaps were not included. Numbers over the branches represent bootstrap coefficients from 1,000 replicas.

FIG. 6.

α-l-Arabinofuranosidase gene transcription in the parental strain QM9414 (a), strains deleted in the first two steps of the l-arabinose pathway (Δxyl1 and Δlad1) (6), and strains deleted in the regulator XYR1 (Δxyr1) or expressing xyl1 constitutively in an XYR1-negative background (Δxyr1 and Ptef1-xyl1) (c). A total of 20 μg of total RNA was loaded per lane, and α-³²P-radiolabeled PCR fragments of the different genes were used as probes.

FIG. 7.

Occurrence of the XYR1-binding consensus GGC(T/A)₄ (15) in the 5′ upstream region of abf1, abf2, abf3, and bxl1. The presence of the consensus is indicated by an arrow (not drawn to scale). Gray arrows indicate the presence of the motif on the sense strand, and open arrows indicate the motif on the antisense strand. The sequences of the two connected motifs are shown below.