Mutation of the Xylanase regulator 1 causes a glucose blind hydrolase expressing phenotype in industrially used Trichoderma strains
© Derntl et al.; licensee BioMed Central Ltd. 2013
Received: 13 December 2012
Accepted: 23 April 2013
Published: 2 May 2013
Trichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation.
Here, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a.
Engineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.
KeywordsTrichoderma reesei Hypocrea jecorina Cellulases and hemicellulases Inducer-independent enzyme production Biofuel Xylanase regulator 1 (Xyr1) Glucose response domain
Trichoderma reesei (telomorph, Hyprocrea jecorina)  is a filamentous ascomycete thriving as a saprophyte on dead plant material. It degrades cellulose and hemicelluloses by secreting a wide array of cellulases and hemicellulases. A genome-wide analysis revealed 10 celluloytic and 16 xylanolytic enzyme-encoding genes in T. reesei. The most abundantly secreted and industrially interesting enzymes are the two main cellobiohydrolases, CBHI and CBHII (EC 18.104.22.168) , and two major specific endo-ß-1,4-xylanases, XYNI and XYNII (EC.22.214.171.124) . We will use the term major, industrially relevant hemicellulases and cellulases (MIHCs) throughout this publication for these two cellulases and two hemicellulases.
The MIHCs work together with further enzymes to degrade cellulose and xylan. This results in the formation of soluble oligo- and monosaccharides, such as cellobiose, D-glucose, xylobiose, and D-xylose. In addition, sophorose is a product of the transglycosylation activity of some of these enzymes . All of these molecules were reported to have influence on the expression of MIHCs in T. reesei. The presence of D-glucose causes carbon catabolite repression (CCR), which results in the secretion of low quantities of MIHCs; expression of XYNI is even completely shut off. Sophorose is the most potent inducer for the expression of CBHI and CBHII. It also triggers the expression of XYNII (reviewed by ). D-xylose modulates XYNI and XYNII expression in a concentration-dependent manner. Strongest induction occurs by using low concentrations (0.5 mM), whereas high concentrations lead to a repressing effect on xylanase expression .
Despite the different patter of inducibility of the expression of MIHCs, it generally depends on the presence of the main transactivator of hydrolases Xyr1 (Xylanase regulator 1). Xyr1 has a Gal4-like Zn2Cys6 binuclear cluster domain, which is involved in DNA-binding. A xyr1 deletion mutant does not produce any MIHCs at the level of either transcription or of protein formation [8, 9]. It has been reported that using D-xylose or xylobiose could not induce the expression of xyr1 itself, even if these saccharides are potent inducers of XYNI and XYNII expression mediated via Xyr1. However, analogous to its target genes, Xyr1 expression is regulated by CCR mediated by Cre1 . Cre1 is a wide-domain regulator that binds under repressing conductions (high concentrations of easily utilisable monosaccharides such as D-glucose or D-xylose) to its binding site in the promoter of e.g. xyr1 or xyn1 resulting in a down-regulation or a complete shut-off of transcription, respectively [11–13].
It is evident that a release from CCR is a useful prerequisite for industrial exploitation of T. reesei for the production of MIHCs. Therefore, a prominent Cre1-deficient mutant strain of T. reesei, Rut-C30, which was described as a high yielding cellulase mutant , has been used as the progenitor strain for many recent industrial strains. However, industrially satisfying production of MIHCs by Rut-C30 and its industrially used offspring still depends on potent induction. Using certain inducing compounds or a particular media composition is the common way to achieve this induction, but both add cost which may lead to a higher price for the resulting enzyme products. In particular, the economical, ecological and socio-economical success of products such as second-generation biofuel strongly depend on their cheap availability of MIHCs as well as a production process using non-food biomass as starting material.
In this study we report on an industrially used T. reesei strain that produces high amounts of MIHCs independent of the presence of a certain inducer. Moreover, this strain shows a glucose-blind phenotype when it comes to expression of MIHCs. Consequently, we analysed this strain at the transcriptional level in order to identify the molecular mechanisms behind this phenotype. Interestingly, we found aside to other mutations a single point mutation in Xyr1 and investigated to which extent this is responsible for the outstanding phenotype of that strain.
A two-step mutant derived from T. reesei Rut-C30 yields elevated and aberrant xylanase expression due to a single point mutation in xyr1
Strong deregulation of expression of MIHCs can be observed in T. reesei Iogen-M8
The outstanding properties of Iogen-M8 with regard to xylanolytic activity were investigated on the level of transcription to identify changes in regulatory mechanisms of expression of MIHCs compared to Rut-C30, its first ancestor with a Cre1-negative background. Therefore, both strains were pre-grown in Mandels-Andreotti (MA) medium containing solely glycerol as carbon source. These mycelia were replaced into MA medium containing either 50 mM D-glucose, 0.5 mM D-xylose, 66 mM D-xylose, or 1.5 mM sophorose as carbon sources or inducers [7, 15, 16]. An additional culture was incubated in MA medium without carbon source as reference. Samples were taken directly from the pre-culture (before the replacement) and after three and six hours of incubation. RNA of these samples was extracted and used as template for RT-qPCR analysis.
In addition, the transcript levels of the main cellobiohydrolase-encoding genes, cbh1 and cbh2, showed a striking increase (up to 10,000-fold) in Iogen-M8 compared to Rut-C30 (compare Figure 2C, D to Figure 2H, I). These genes remain subject to induction in Iogen-M8 as a comparison of their transcript levels derived from incubation without carbon source to those with sophorose reveals (Figure 2C, D), even if the extent of inducibility is less pronounced than in Rut-C30 (Figure 2H, I). Notably, in both strains xyr1 transcription is inducible by sophorose (Figure 2E, J), and the patterns of transcript levels of cbh1 and cbh2 reflect those of xyr1 on all carbon sources or inducers tested (Figure 2C-E and Figure 2H-J).
In summary, Iogen-M8 has general highly elevated transcript levels of all genes under all investigated conditions compared to Rut-C30. Consequently, we conclude Iogen-M8 exhibits a strong deregulation in expression of MIHCs.
Deregulation of MIHCs expression only partly occurs in the parental strain of Iogen-M8
A824V transition in Xyr1 causes strong deregulation of xylanase expression and elevated basal cellulase expression
In accordance with all other strains we could observe that xyr1, cbh1, and cbh2 are inducible by sophorose in Iogen-M4X (Figure 4C-E). As a consequence of this observation, we questioned if this is due to a principle regulatory mechanism also present in the wild-type QM6a and not restricted to the strains investigated in this study so far (i.e., a consequence of mutagenesis and selection).
Gene expression of cbh1 and cbh2 strictly depends on the level of xyr1 transcription
Notably, transcript levels of xyr1 are induced on sophorose in all strains investigated in this study so far (Figure 2E, J, Figure 3E, and Figure 4E). Moreover, the patterns of transcript levels of cbh1 and cbh2 strictly reflect those of xyr1 under all conditions tested (Figure 2C-E, Figure 2H-J, Figure 3C-E, and Figure 4C-E). As mentioned, all these strains are derivatives of Rut-C30, a mutant derived from the wild-type strain QM6a (Figure 1A). We questioned whether the correlation between xyr1 transcript levels and those of cbh1 and cbh2 can already be observed in the wild-type strain and performed an analogous carbon source replacement experiment using QM6a.
In silico characterization of the xyr1 sequence part that bears the A824V transition
According to DELTA-BLAST (http://www.ncbi.nlm.nih.gov/), Xyr1 has a so-called fungal transcription factor regulatory middle homology region (FTFRMH region) (from L359 to L904, cd12148 with an E-value of 1.77e-11) located next to the Gal4-like DNA-binding domain (from R93 to Y126, cd00067 with an E-value of 2.81e-10). The A824V mutation of Xyr1 is located within the FTFRMH region. This FTFRMH region is present in the large family of fungal zinc cluster transcription factors that contain an N-terminal GAL4-like Cys6 zinc binuclear cluster DNA-binding domain . The C-terminal domain of Cep3p, a subunit of the yeast centromere-binding factor 3, is similar to the FTFRMH region (E-value 1.22e-04). A 3D model is available for a great part of Cep3p based on X-ray diffraction . An alignment of the FTFRMH regions of Xyr1, Cep3p, and the consensus sequence matches position A824 of Xyr1 with I463 of Cep3p (Constraint-based Multiple Alignment Tool, http://www.ncbi.nlm.nih.gov/) and is shown in an additional file [see Additional file 3]. I463 of Cep3p is located in the middle of an α-helix, which reaches from M458 to I475. A graphic display is given in an additional file (see Additional file 4). Additionally, three different secondary structure predictions for the domain of Xyr1 locate A824 in the middle of an α-helix (http://www.compbio.dundee.ac.uk/, BCL::Jufo9D at http://meilerlab.org/, http://bioinf.cs.ucl.ac.uk/psipred/). Consequently, we assume that the A824V mutation in Xyr1 possibly leads to a change in secondary structure.
During this study we found that the expression of the two major cellulase genes cbh1 and cbh2 strictly follow xyr1 transcript levels. Accordingly, Portnoy and co-workers reported that in a cellulase-overproducing strain, xyr1 transcript levels are elevated compared to common T. reesei strains (as QM9414) . These findings suggest that cellulase gene expression is highly dependent on the amount of Xyr1.
Otherwise, we found that the expression of the two major xylanases, xyn1 and xyn2, does not strictly follow xyr1 transcript levels. Interestingly, in Aspergillus nidulans a constitutive expression of xlnR (the xyr1 homolog ) under the gpdA promoter led to enhanced and continuously high xlnA/B transcript formation, while xlnD transcript diminished after 1 h and did not follow the xlnR transcript level pattern anymore . Altogether, we assume that regulation of xylanase gene expression is not directly dependent on the amount of Xyr1 and seems to rely on additional mechanisms.
The different Xyr1 responsiveness of cellulases and xylanases was also observed in a T. reesei QM9414 strain constitutively expressing xyr1. There, the cellulolytic regulon of Xyr1 was positively affected, while the xylanolytic regulon was negatively affected . Notably, this observations are supported by the number of in silico identified Xyr1-binding sites in respective promoter regions. The reported Xyr1-binding site, 5’-GGC(T/A)3-3’ [23–26], occurs 14 times within 1 kb of the promoter region of cbh1, whereas it occurs only 4 times within 1 kb of the promoter region of xyn1. Currently ongoing in vivo footprinting analysis of corresponding promoters revealed that 12 and 2 of these sites are differently contacted comparing inducing and repressing conditions, respectively (unpublished observations, Gorsche, R., Lichti, J., Mach, R.L., Mach-Aigner, A.R.). Supportively, we found during this study that sophorose, which has been known for decades as a potent cellulase inducer , positively influences xyr1 expression. As stated before, expression of the two major cellulase genes cbh1 and cbh2 strictly follow xyr1 transcript levels. Taken together, we assume, that induction of cbh1 and cbh2 by sophorose is a direct result of elevated xyr1 transcription levels. The issues discussed so far could be observed in all of the T. reesei strains investigated in this study, including the wild-type QM6a.
However, two outstanding phenomena could be observed in Iogen-M8: first, a strong deregulation of xylanase expression and second, a generally very high level of transcript formation of MIHCs. By investigating correspondingly manipulated strains, we found that a single point mutation in Xyr1 (A824V) is fully responsible for the deregulation of xylanase gene expression and the high basal level of cbh1 and cbh2 expression. A similar phenomenon was briefly described for the XlnR in A. niger. A V756F mutation resulted in constant xylanase activity even under repressing conditions . Nonetheless, we currently cannot provide a mechanistic explanation, it is noteworthy that the A824V transition is located in a predicted α-helix within a FTFRMH region. As alanine has the lowest helix propensity (0 kcal/mol), whereas valine has a higher value (0.61 kcal/mol) . The mutation in Xyr1 may result in a structural change. Notably, the previously mentioned V756 in XlnR corresponds to V821 in Xyr1 located in the same predicted α-helix as A824.
For Gal4 it was reported that glucose has a direct effect on its activity. The localisation of the glucose response domain in Gal4 was narrowed down to a central region , in which the FTFRMH region lies (E-value 4.57e-50). Albeit a functional similarity of both regions seems likely, we found that the phenotype of Iogen-M8 is not linked just to D-glucose. Consequently, we presume that the corresponding domain of Xyr1 is a more generally regulatory region.
We have shown that a single point mutation in a regulatory domain of the central regulator Xyr1 has tremendous effects on expression behaviour of MIHCs in an industrially used strain of T. reesei. We believe that this finding is a very promising starting point for directed strain developments by e.g. transcription factor engineering. Supporting results from A. niger suggest that the observed phenomenon is not limited to Trichoderma. Therefore, we recommend manipulations of the regulatory domain of this group of Gal4-like transcription factors as a strategy for inducer-independent expression of MIHCs.
The following T. reesei strains were used throughout this study: the wild-type strain QM6a (ATCC 13631), Rut-C30, which was described as a high yielding cellulase mutant of QM6a (ATCC 56765) , Iogen-M4, which is a spontaneous mutant of Rut-C30 , Iogen-M8, which is a strain obtained by UV mutation from Iogen-M4, Iogen-M4X, which is a derivate of Iogen-M4 bearing an introduced point mutation (A824V) in Xyr1, and Iogen-M8X, which is a derivate of Iogen-M8 bearing a reconstituted wild-type xyr1. All strains were maintained on malt extract agar or potato-dextrose-agar.
UV mutagenesis and screening
In order to obtain Iogen-M8, conidia of Iogen-M4 from a single potato dextrose plate were suspended in 10 ml of sterile distilled water and filtered through glass wool to remove any mycelia. The conidia were diluted in water to a concentration of 105 per mL and irradiated in a thin film with a germicidal lamp at a distance of 7 cm. Irradiation for 90–120 seconds was generally sufficient to give 1 - 10% survival. Diluted suspensions were plated onto selective media containing acid swollen cellulose as the primary carbon source and 4 g L-1 of the glucose anti-metabolite 2-deoxyglucose. Iogen-M8 was selected for its ability to grow and produce large clearing zones on this medium, indicative of hyperproduction of cellulose-degrading enzymes.
For carbon source replacement experiments, mycelia were pre-cultured in 1 L Erlenmeyer flasks on a rotary shaker (180 rpm) at 30°C for 18 h in 300 mL of Mandels-Andreotti (MA) medium  containing 105 mM of glycerol as the sole carbon source. A total of 109 conidia per liter (final concentration) was used as the inoculum. Pre-grown mycelia were washed, and equal amounts were resuspended in MA media containing D-xylose, D-glucose, and sophorose in concentrations as stated. Mycelia were also grown in MA media without any carbon source (control). Samples were taken directly before the carbon source replacement (after harvesting the mycelia after pre-growth), after 3 hours, and after 6 hours of incubation. Samples were derived from three biological replicates and were pooled before RNA extraction.
Cultivations in a bioreactor were run in a 14 L pilot scale fermentation vessel (Model MF114 New Brunswick Scientific Co.) set up with 10 L of Initial Pilot Media. Operational parameters were: agitation at 500 rpm, air sparging at 8 standard L min-1, a temperature of 28°C, and pH was maintained at 4.0 - 4.5 during batch growth and pH 5.0 during enzyme production. An additional file provides a more detailed bioreactor protocol (see Additional file 5).
Growth on xylan plates was performed using MA medium containing 0.2% (w/v) azo-xylan (Megazym, Wicklow, Ireland) at 30°C.
Determining the relative concentrations of cellulases and hemicellulases
The relative concentrations of cellulases and hemicellulase mixtures in the culture supernatants produced in bioreactors were determined by ELISA. Supernatants and purified component standards were diluted 1–100 μg mL-1 in phosphate-buffered saline, pH 7.2 (PBS) and incubated overnight at 4°C in microtitre plates. The plates were washed with PBS containing 0.1% Tween 20 (PBS/Tween) and incubated in PBS containing 1% BSA (PBS/BSA) for 1 h at room temperature (RT) followed by washing with PBS/Tween. Rabbit polyclonal antisera specific for CBHI, CBHII, EGI, XYNI, XYNII, and BGLI were diluted in PBS/BSA, added to separate microtitre plates and incubated for 2 h at RT. Plates were washed and incubated with a goat anti-rabbit antibody coupled to horseradish peroxidase for 1 h at RT. After washing, tetramethylbenzidine was added and incubated for 1 h at RT. The absorbance at 660 nm was measured and converted into protein concentration using the standards. The relative concentration refers to the total protein concentration of the culture supernatants.
Isolation of chromosomal DNA and genome sequencing
After growth in 75 mL of MA medium containing 50 mM glucose at 28°C for 4 days, cultures were filtered through sterile glass fibre filters and frozen in liquid nitrogen. Biomass was ground to fine powder and resuspended in 30 mL of lysis buffer (20 mM EDTA, 10 mM Tris–HCl, pH 7.9, 1% Triton ×-100, 500 mM guanidine-HCl, 200 mM NaCl, 0.76 mg mL-1 Driselase® 0.4 mg mL-1 T. harzianum beta-glucanase, and 0.8 μg mL-1 T. viride chitinase C). After treatment with RNase A and RNase T1 at 20 μg mL-1 and 100 U mL-1 final concentrations (50°C, 1 h), Proteinase K was added to a concentration of 0.8 mg mL-1 (50°C, 1 h). Following centrifugation (20 min at 12,000 × g), the clarified lysate was used to isolate chromosomal DNA using the Qiagen® Genomic-tip 500/G genomic DNA isolation kit (Qiagen Inc.-Canada, Ontario, CA).
Genomic DNA was sequenced using the Illumina/Solexa GAIIx sequencing technology (as distributed by Montreal Biotech Inc., Quebec, Canada) utilizing two lanes per strain (one lane of single read and one lane of paired-end reads). The raw sequences were assembled directly against publicly available sequence for strain QM6a (http://genome.jgi-psf.org/Trire2/Trire2.info.html) using DNAstar Seqman NGen® software (DNASTAR Inc., Wisconsin, USA). After assembly, a single nucleotide polymorphism calling procedure was used to identify a table of high-confidence sequence variants.
Oligonucleotides used in this study
Sequence (5′ - 3′)
xyr1 3′-UTR cloning
xyr1 5′-UTR and coding sequence cloning
cbh1 3′-UTR cloning
Separately, 4.1 kb fragments were amplified from Iogen-M4 and Iogen-M8 gDNA using primers FT165f and FT166r comprising the xyr1 5’-UTR and the wild-type xyr1 or xyr1(A824V) coding sequence, respectively. The resulting products were used as templates for a second PCR using primers FT167f and FT166r. FT167f introduces Xba I and Pac I sites at the 5’-end of the amplified products. These PCR products were used as templates for a third PCR using primers FT168r and FT169f, to generate fragments suitable for subsequent recombination steps.
A 586 bp fragment comprising the 3’-UTR of the cbh1 gene was amplified from Iogen-M4 gDNA using primers FT170f and FT171r.
pSC1 was linearized using Nhe I and Not I and recombined with the 4.1 kb fragment containing the xyr1 5’-UTR and xyr1 or xyr1(A824V) coding sequence and the 586 bp cbh1 3’-UTR fragment using the In-Fusion HD Cloning System (Clontech). This resulted in pSCxyr1-TV and pSCxyr1A824V-TV used for fungal transformation. Vector maps are provided in an additional file (see Additional file 6).
The protoplast transformation of T. reesei was performed as described in U.S. Patent No. 8,323,931. To obtain Iogen-M4X, the plasmid pSCxyr1A824V-TV was transformed into a uridine auxotroph of Iogen-M4 selecting for uridine prototrophy on modified (MA) medium . Introduction of pSCxyr1-TV into a uridine auxotroph of Iogen-M8, followed by selection for uridine prototrophy, resulted in a mutant strain bearing the wild-type xyr1 namely Iogen-M8X.
RNA-extraction and reverse transcription
Harvested mycelia were homogenized in 1 mL of peqGOLD TriFast DNA/RNA/protein purification system reagent (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, US). RNA was isolated according to the manufacturer’s instructions, and the concentration was measured using the NanoDrop 1000 (Thermo Scientific, Waltham, US).
Synthesis of cDNA from mRNA was carried out using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions.
Quantitative PCR analysis
Quantitative PCRs were performed in a Mastercycler® ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicate. The amplification mixture (final volume 25 μL) contained 12.5 μL 2 × iQ SYBR Green Mix (Bio-Rad Laboratories, Hercules, USA), 100 nM forward and reverse primer and 2.5 μL cDNA (diluted 1:100). Primer sequences are provided in Table 1. Cycling conditions and control reactions were performed as described previously . Data normalization using sar1 and act as reference genes, and calculations were performed as published previously . The transcript levels in all figures were referred to those from QM6a incubated without carbon source for 3 h; therefore, they can be compared cross-figure wisely.
Carbon catabolite repression
Fungal transcription factor regulatory middle homology region
Major, industrially relevant hemicellulases and cellulases
Bovine serum albumine
Xylanase regulator 1.
This study was supported by Iogen Energy Corporation and by two grants from the Austrian Science Fund (FWF): [P20192, P24851] given to R.L.M and A.R.M.-A., respectively.
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