Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains

Abstract

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V _max = 2646 nkat/mg with a K _m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Keywords: Biofuels; Biorefinery; GH11 family; Recombinant protein production; Trichoderma reesei; XYN1; Xylanase.

Fig. 1

Alignment of the first 100 nucleotides of xyn5 coding region of different Trichoderma spp. and Trichoderma reesei wild-type strains. Nucleotides were aligned and differences were manually annotated. When translated from the conserved start ATG found in all xyn5 genes, the ORF of T. reesei QM6a misses 4 bp which leads to a frameshift and the termination of the ORF by a TAA stop codon. The start codon of the original NCBI database entry (XP_006969745.1) is found 46 bp downstream which misses therefore the XYN5 signal peptide sequence. GenBank accession numbers for xyn5 are KX455497 (T. reesei QM6a), KX139139 (T. reesei TUCIM1282). The other sequences were retrieved from the JGI Genome Portal MycoCosm (http://genome.jgi.doe.gov/programs/fungi/index.jsf) and are Trias1|83211 (Trichoderma asperellum), Triat2|46014 (Trichoderma atroviride), TriviGv29_8_2|65505 (Trichoderma virens), and Triha1|118868 (Trichoderma harzianum). Residues highlighted by a black background are conserved in at least 90%, while residues highlighted by gray in 40% of the xyn5 sequences

Fig. 2

Transcriptional regulation of xyn5 in T. reesei TUCIM1282 (violet bars) and QM6a (red bars). Strains were grown for 24 h on 1% (v/v) glycerol as carbon source and then transferred to medium with 1 mM d-xylose (a) and 1 mM l -arabinose (b). The fold change of xyn5 expression in QM6a and TUCIM1282 was measured 2, 4 and 6 h after transfer to the inducing medium and normalized to the expression of tef1. Xyn5 transcript levels were compared to the corresponding time points of cultures without a carbon source. Values represent the mean ± SD of at least two biological replicates (color figure online)

Fig. 3

3D surface model of XYN5 from T. reesei TUCIM1282 based on the structure of XYN1 (PDB: 1XYN). The protein is primarily composed of GH11 typical antiparallel β-sheets with a unique α-helix. These secondary structures form a substrate binding cleft, which is illustrated on the left side. The two GH typical catalytic residues, E75 and E164, are located within this cleft (right side)

Fig. 4

SDS-PAGE of the purified and deglycosylated XYN5 expressed in T. reesei strain xyn5g1. Following purification of XYN5 by cation exchange chromatography, two bands corresponding to T. reesei TUCIM1282 XYN5 were detected. This double band was reduced to a single band by Endo-T treatment of the XYN5 fraction. Lanes: 1, Endo-T; 2, molecular size marker; 3, purified XYN5 protein (2 μg) after cation exchange chromatography; 4, Endo-T treated purified XYN5 (2 μg)

Fig. 5

pH and temperature optimum of the recombinantly expressed XYN5 from T. reesei TUCIM1282 in T. reesei Δxyr1. For determination of the pH optimum, beechwood xylan was dissolved in 0.1 M McIlvaine buffer and DNS-based assays were performed at 50 °C. For the determination of the temperature optimum, the substrate was dissolved in 10 mM NaAc buffer pH 4. Highest enzyme activity was detected at pH 4 and 50 °C. Mean ± SD of two biological replicates are shown

Fig. 6

Increase in xylanolytic activity upon homologous replacement of the non-functional xyn5 in T. reesei strain Δtku70 by the T. reesei TUCIM1282 xyn5. Total xylanase activity of the strains harboring the full-length xyn5 (blue line) was compared to the parental strain Δtku70 (red line) during growth on MA medium using beechwood xylan, lactose, or wheat straw as carbon source. Lactose and beechwood xylan samples represent the activities corrected for biomass, whereas wheat straw samples represent volumetric activities. Mean ± SD of two biological replicates is shown (color figure online)