A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation

AbstractHypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus

d-Xylose as a Repressor or Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)▿

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

A long noncoding RNA promotes cellulase expression in Trichoderma reesei

Abstract Background Due to its capability to secrete large quantities of plant biomass degrading enzymes (PBDE), Trichoderma reesei is widely applied for industrial purposes. In nature, expression of PBDE is efficiently regulated in this fungus. Several factors involved in this regulatory network have been identified. However, most of them are transcription factors. Long noncoding RNAs (lncRNAs) emerged as common players acting on epigenetic or transcriptional regulation in several eukaryotic organisms. To date, no lncRNA has been described in filamentous fungi. Results A lncRNA termed HAX1 was identified in T. reesei QM9414. In this study, it was characterized and evidence for its regulatory impact on cellulase expression was provided. Interestingly, different versions of HAX1 were identified in different strains (namely, QM6a, QM9414, and Rut-C30), varying in terms of RNA length. Remarkably, considerable longer variants of this lncRNA are present in hypercellulolytic strains compared to the wild-type strain QM6a. Based on these results, a correlation between RNA length and the functional impact of HAX1 on PBDE expression was supposed. This assumption was verified by overexpressing the most abundant HAX1 versions identified in QM6a, QM9414, and Rut-C30. Such HAX1 overexpression on the one hand was suitable for regaining the function in hax1 disruption strains, and on the other hand resulted in notably higher cellulase activities in QM6a, especially by the expression of longer HAX1 versions. Conclusion With HAX1, for the first time the regulatory role of a lncRNA in filamentous fungi was uncovered. Besides this, a new player involved in the complex regulation of PBDE expression in T. reesei was identified. Due to its enhancing effect on cellulase activity, HAX1 was shown to be not only interesting for basic research, but also a promising candidate for expanding the set of biotechnological tools for industrial application of T. reesei

MOESM5 of A long noncoding RNA promotes cellulase expression in Trichoderma reesei

Additional file 5. RNA stability of the three HAX1 versions. The T. reesei strains QM6a (blue), QM9414 (yellow) and Rut-C30 (purple) were grown in MA medium containing 1% (w/v) α-d-lactose for 24 h and transcription was inhibited by the addition of DRB. Samples were taken before the addition of DRB (reference sample) and 30 min, 60 min, 90 min and 120 min after the addition of DRB. RT-qPCR was performed using cDNA from QM6a for the amplification of hax1QM6a (primer pair up-hax1 for_2 and hax1 rev_1.Intron, blue), from QM9414 for the amplification of hax1QM9414 (primer pair hax1 for_qPCR_QM9414 and hax1 rev_up-Intron; yellow) and from Rut-C30 for the amplification of hax1Rut-C30 (primer pair up-hax1 for_1 and hax1 rev_up-Intron, purple). Transcript levels were normalized to act and sar1, refer to the sample taken before the addition of DRB (time point 0 min) and are given in logarithmic scale (lg). All standard deviations of technical triplicates are < 0.06

MOESM6 of A long noncoding RNA promotes cellulase expression in Trichoderma reesei

Additional file 6. RT-qPCR of the three hax1 versions in QM9414 disruption strains. Analysis of the formation of hax1 transcripts, i.e., hax1QM6a (graph on top), hax1QM9414 (graph in the middle) and hax1Rut-C30 (graph at the bottom), in QM9414_Dhax1_8 (green) and QM9414_Dhax1_14 (blue) and their parent strain QM9414 as a positive control (grey). The cDNAs used as templates were derived from cultivation on lactose for 48 h (QM9414_Dhax1 strains) or replacement to medium without carbon source (positive control). For amplification the primer pairs up-hax1 for_2 and hax1 rev_1.Intron (hax1QM6a), hax1 for_qPCR_QM9414 and hax1 rev_up-Intron (hax1QM9414), or up-hax1 for_1 and hax1 rev_up-Intron (hax1Rut-C30) were used. Analysis was performed in technical triplicates. A no template control (red) and a negative control (yellow) were included in each run. Measured fluorescence is plotted against the number PCR cycles

MOESM1 of A long noncoding RNA promotes cellulase expression in Trichoderma reesei

Additional file 1. Transcript levels of the neighbouring genes of the targeted site for amdS integration. RT-qPCR results of an undescribed protein referred to as hypothetical protein (Protein ID 108999) (A) and the 2-isopropylmalate synthase (Protein ID 79495) (B) in T. reesei strains QM9414 (PS; black bars), QM9414_Dhax1_8 (D8; orange bars) and QM9414_Dhax1_10 (D10; red bars). cDNAs used as templates were derived from cultures that were pre-grown and transferred to medium without carbon source, 1% (w/v) d-glucose (G) or 1.5 mM sophorose (S) for 3 h. Transcript levels were normalized to act and sar1, refer to QM9414 (no carbon source) and are given in logarithmic scale (lg). Analysis was performed in technical triplicates. The error bars depict the standard deviation and different letters denote statistical difference among compared data employing ANOVA (P < 0.05)

MOESM2 of A long noncoding RNA promotes cellulase expression in Trichoderma reesei

Additional file 2. Codon usage of the predicted hax1 gene. For each of the 64 codons potentially making up a protein the following information is listed: base triplet; the encoded amino acid (given as one-letter code); fraction out of the 313 codons constituting hax1; frequency per thousand; absolute number of occurrence (from a total of 313 codons). Asterisks indicate stop codons