key: cord-0048339-3t3iezi5
authors: Derntl, Christian; Mach, Robert; Mach-Aigner, Astrid
title: Application of the human estrogen receptor within a synthetic transcription factor in Trichoderma reesei
date: 2020-08-03
journal: Fungal Biol Biotechnol
DOI: 10.1186/s40694-020-00102-3
sha: 4257b0b9b7c4fd45ea55ed81933f2166d8fbcb4c
doc_id: 48339
cord_uid: 3t3iezi5

BACKGROUND: Synthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial applications. The human estrogen receptor α contains a ligand binding domain that enforces dimerization and nuclear import upon binding of the inducer 17β-estradiol. In this study, we tested the potential of this ligand binding domain to be used in filamentous fungi as an auto-regulatory domain in a synthetic transcription factor. RESULTS: We constructed the synthetic transcription factor SynX by fusing the DNA-binding domain of Xyr1 (Xylanase Regulator 1), the transactivation domain of Ypr1 (Yellow Pigment Regulator 1), and the ligand binding domain of the human estrogen receptor α. SynX is able to strongly induce the gene expression of xylanases and an aldose reductase by addition of 17β-estradiol, but SynX does not induce gene expression of cellulases. Importantly, the induction of xylanase activities is mostly carbon source independent and can be fine-tuned by controlling the concentration of 17β-estradiol. CONCLUSION: The ability of SynX to induce gene expression of xylanase encoding genes by addition of 17β-estradiol demonstrates that the ligand binding domain of the human estrogen receptor α works in filamentous fungi, and that it can be combined with a transactivation domain other than the commonly used transactivation domain of herpes simplex virion protein VP16.

Controlled induction of gene expression is a necessity for basic research and industrial applications. An ideal expression system has no basal expression and can be induced by a stimulus that does not interfere with the native metabolism or regulatory network. Moreover, the expression should be tunable and reach a high expression level. Such a system enables the expression of genes of interest at a desired time point to desired intensities, allowing for example the expression of toxic or harmful proteins or the synthesis of toxic substances.

For filamentous fungi several such synthetic expression systems were developed previously. In 2005, the human estrogen receptor α (hERα) was used successfully in A. niger and A. nidulans and could drive gene expression from a synthetic promoter upon induction with estrogenic substances [1] . The hERα is a nuclear receptor reacting to a group of small, hydrophilic substances, including the hormone 17β-estradiol. The hERα consists of 5 structural domains (A/B, C, D, E, and F). The A/B domain supports the transactivation activity of domain E, Domain C is the DNA-binding domain (DBD), domain D is a linker containing a nuclear localization signal, domain E is the ligand binding domain (LBD), and domain F appears to modulate the transcriptional activity, co-activator interactions, dimerization, and stability of the receptor [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . Upon binding of an inducer to the LBD, hERα undergoes a conformational change, which results in dimer stabilization, nuclear import, and exposure of surfaces for interactions with the basic transcription machinery (the LBD is also responsible for transactivation) [6, 7, 10, 13, 14] . Refer to the review by Yaşar et al. [15] for a summary about the current knowledge on the hERα.

Also in 2005, the Tet-on/Tet-off system, which had previously been successfully applied in several eukaryotic species [16] [17] [18] [19] [20] [21] , was demonstrated to work in A. fumigatus [22] . This system is based on the bacterial TetR repressor that regulates the tetracycline resistance operon in Escherichia coli by binding to the tetO sequence upon the presence of tetracycline or doxycycline. For applications in eukaryotes, TetR was fused to the transactivation domain (TAD) of herpes simplex virus protein VP16 and a synthetic promoter constructed by adding the tetO sequence to a minimal promoter [23] . This system was further optimized for application in A. niger [24] [25] [26] and was also successfully applied in Fusarium fujikuroi [27] .

For Trichoderma reesei two inducible expression systems responding to light [28] or copper [29] were developed. Notably, the inducers also affect the basic biology of T. reesei and do not allow the exclusive induction of the target gene(s). In another report, the application of a synthetic expression system that is suitable for a broad range of fungal species including T. reesei was described [30] . This system consists of a synthetic transcription factor (TF) and a set of differently strong promoters allowing the expression of gene(s) of interest at different constitutive levels. The synthetic TF itself is a fusion of the DNAbinding protein Bm3R1 from Bacillus megaterium, the SV40 nuclear localization signal, and the TAD of VP16 [30] . Please refer to the review by Kluge et al. [31] for a more detailed and comprehensive summary of inducible expression system in filamentous fungi.

In T. reesei, the Gal4-like TF Xyr1 (Xylanase regulator 1) is the main activator for the expression of most cellulases and hemicellulases [32] . The main cellulases are the two cellobiohydrolases CBHI and CBHII (EC 3.2.1.91) and the endo-glucanase EGLI (EC 3.2.1.4) [33] . The major hemicellulases are the two endo-ß-1,4-xylanases XYNI and XYNII (EC 3.2.1.8) [34] . Xyr1 is additionally essential for the expression of the aldose reductase Xyl1 (EC 1.1.1.307), which catalyzes the first reaction in the catabolism of certain monosaccharides, such as xylose, galactose, and arabinose [35, 36] .

Despite being regulated by the same main transactivator, the expression of cellulases and xylanases is induced under different conditions. Simplified, the expression of the cellulases is induced on lactose, cellulose, and sophorose (transglycosylation product of cellobiose), whereas the expression of the xylanases is induced on xylan and low concentrations of xylose, and partially on lactose [37] .

The expression of Xyr1 itself is down-regulated under carbon catabolite repression (CCR), mediated by Cre1 [38, 39] and is induced on cellulase-inducing conditions [40] [41] [42] . Recent publications suggest the transcription factors Ace3 and Rxe1 to be involved in the induction of Xyr1 expression [43] [44] [45] . Notably, the expression of the Xyr1 target genes is also regulated by further factors and mechanisms, such as the TFs Ace1 [46] , Ace2 [47] , Ace3 [43] , Xpp1 [48] and Rce1 [49] , the mating type locus protein Mat1-2-1 [50] , the photoreceptor Env1 [51] , the protein methyltransferase Lae1 [52] , the velvet complex protein Vel1 [53] , the Hap2/3/5 complex [54] [55] [56] , and the MAP kinases Tmk2 [57] and Tmk3 [58] .

In a recent study, we constructed a fusion transcription factor (TF), termed XY1, which consists of the N-terminus of Xyr1 and the C-terminus of Ypr1 (Yellow pigment regulator 1) [59], the main activator of sorbicillinoid biosynthesis [60] . Bearing the DBD of Xyr1, the fusion TF XY1 is able to induce the expression of the Xyr1 target genes [59] . The C-terminus of Ypr1 contains the so-called fungal transcription factor middle homology region (FTFMHR), which is responsible for the transactivation [59] . Overexpression of the fusion TF induced the expression of cellulases and xylanases in the Xyr1-deficient recipient strain Xyr1′(81) on different carbon sources, even on d-glucose [59] . The transcript levels of the main xylanase genes xyn1 and xyn2, and the aldose reductase gene xyl1 reached remarkable high levels and were completely deregulated regarding the carbon source used [59] .

In this study we describe the construction of a synthetic TF with the aim to enable controllable expression of the Xyr1 regulon in T. reesei. We fused the DBD of Xyr1, the TAD of Ypr1, and the LBD of hERα to create the TF SynX. A constitutive expression cassette for the SynX was inserted into a Xyr1-deficient strain. The tightness of the expression system and the ability of the SynX to induce expression of the main Xyr1 target genes were determined on transcript and enzymatic levels; further we studied the influence of different carbon sources and different concentrations of estradiol on the xylanolytic and cellulolytic activities.

In order to use the hERα in a synthetic transcription factor, the inducing substance 17β-estradiol must not influence the basic biology of the host organism at working concentrations. Consequently, we performed a growth experiment in order to estimate to which extend T. reesei can be exposed to 17β-estradiol. The wild-type like strain Δtmus53 was cultivated on potato dextrose agar plates in the presence of different concentrations of 17β-estradiol. As a control only the solvent dimethyl sulfoxide (DMSO) was used. After 72 h of growth we observed a growth reduction only in the presence of 10 and 100 µM 17β-estradiol (Fig. 1 ). For comparison, the 17β-estradiol concentration in the female human body ranges from 20 to 500 pg/mL [61] , which corresponds to 0.0734 to 1.835 nM. The hERα in Aspergillus sp. responded to similar concentrations [1] as in the human body. In a previously developed synthetic expression system using the hERα in plants, the standard working concentration of 17β-estradiol was 2 µM, however, the system responded to concentrations as low as 8 nM [62] . We concluded that we could use 17β-estradiol in T. reesei for a synthetic expression system as long as the working concentration stays within the nM or low µM range.

Xyr1′(81) is a Xyr1-deficient strain that is a useful recipient strain for the overexpression of Xyr1 or of fusion TFs containing the Xyr1 DBD. Xyr1′(81) bears a non-sense mutation in Xyr1 at position 81 and a pyr4 deletion, which results in an complete abolishment of xylanolytic and cellulolytic activities [59] . In this study, we inserted the synthetic transcription factor SynX into Xyr1′(81). SynX consists of the DBD of Xyr1 (aa 1-336), the TAD of Ypr1 (aa 185-674), and the C-terminal part of hERα (aa 282-595) which contains a nuclear localization signal and the LBD (Fig. 2) . The mentioned domains of Xyr1 and Ypr1 were previously successfully combined to form the fusion TF XY1 [59]. The indicated part of the hERα was previously successfully used in a fusion TF in plants [62] . For the construction of SynX, the coding sequence for the hERα LBD was codon-optimized for T. reesei (Additional file 1). An overexpression cassette for SynX using the strong, constitutive tef1 promoter and the terminator of cbh2 was inserted into the pyr4 locus by transforming the linearized plasmid pRP4-SynX into strain Xyr1′(81) yielding strain TXYE (Fig. 3a) . The correct and single copy integration of the SynX overexpression cassette into the pyr4 locus was tested by PCR and Southern blot analysis (Fig. 3b, c) .

To test whether SynX can complement for Xyr1-deficiency and induce gene expression of xylanases, we cultivated TXYE, its Xyr1-deficient parent strain Xyr1′(81), and the Xyr1 overexpression strain TX(WT) as positive control on xylan plates. Plates were supplemented with different concentrations of 17β-estradiol dissolved in DMSO and the solvent alone as control (Fig. 4) . After 3 days of cultivation we observed no clearing zone around Xyr1′(81), which indicates the absence of xylanolytic activity, confirming previous results [59] . The Xyr1 overexpression strain TX(WT) produced high levels of xylanolytic activity regardless of the 17β-estradiol concentration. A clearing zone around TXYE was exclusively observed in the presence of 17β-estradiol (Fig. 4) .

Next, we determined the minimal and optimal working concentration of 17β-estradiol and investigated the possibility of the fine-tuning potential of this synthetic Glucose is a repressor of xylanase expression, glycerol is considered to be neutral regarding the expression of xylanases, xylan is a strong natural inducer of xylanase expression, and lactose is a strong inducer of cellulase expression, that can also induce expression of XYNII [55]. We measured the resulting biomass and xylanolytic activity after 48 h of cultivation in shake flasks (Fig. 5) . Notably, no growth was observed on xylan and lactose in the absence of 17β-estradiol (Fig. 5a ). This can be explained by the 17β-estradiol-dependency of SynX.

In wild-type strains, Xyr1 is essential for the expression of the aldose reductase encoding gene xyl1, which is in turn essential for growth on xylan and lactose [32, 36] . In TXYE, the 17β-estradiol-sensing SynX replaces the function of Xyr1. On both carbon sources, growth could be restored by addition of only 3 nM 17β-estradiol (Fig. 5a) . Interestingly, no growth inhibition was observed at high 17β-estradiol concentrations in the shake flask cultivation (Fig. 5a) , in contrast to the observed growth inhibition on plates (Fig. 1) . Regardless of the carbon source used, no xylanolytic activity could be measured in the absence of 17β-estradiol (Fig. 5b) , which is in strict accordance with results from the previously described xylan plate assay (Fig. 4) . Low xylanolytic activity was already detected at 1 nM 17β-estradiol on all carbon sources tested (Fig. 5b) . Further, we observed a 17β-estradiol concentration dependency of the resulting xylanolytic activities. On glucose, the maximum xylanolytic activity was approx. 650 U/g at 3 µM 17β-estradiol, on glycerol approx. 950 U/g at 30 µM, on xylan approx. 2700 U/g at 1 µM, and on lactose approx. 615 U/g at 10 µM 17β-estradiol. On glucose and xylan, high 17β-estradiol concentrations (10 µM and 30 µM) had a negative impact on the resulting xylanolytic activities (Fig. 5b) . We decided to use 300 nM as standard working concentration because on xylan and lactose the measured xylanolytic activities were very close to the respective maximum activity (Fig. 5b) , while on glucose and glycerol the maximum activities were reached at 17β-estradiol concentrations that we considered to be too high for a feasible application.

We performed a similar experiment with the optimal 17β-estradiol concentration of 300 nM in biological triplicates and for a longer cultivation period (72 h) to validate the results from the experiment before (48 h cultivation time, no replicates). Again, we measured no xylanolytic activities in the absence of 17β-estradiol, confirming the tightness of the system (Fig. 6a) . In the presence of 300 nM 17β-estradiol, the xylanolytic activities in the supernatant activities reached 160, 730, 3706, and 239 U/g on glucose, glycerol, xylan, and lactose, respectively (Fig. 6a) . As mentioned previously, Xyr1 is also the main activator for the expression of cellulases in T. reesei. Consequently, we were interested in the influence of SynX on the expression of cellulases. We measured the total cellulolytic activity using resorufin-beta-d-cellobioside as substrate and detected only low cellulolytic activity (Fig. 6b) . On xylan, approx. 8 arbitrary U/g of cellulolytic activity were detected regardless if 17β-estradiol was added or not (Fig. 6b) . This is in the same range as the activity induced by XY1 in our previous study (approx. 10 arbitrary U/g, see [59] ). On lactose, only very low activity was detected, regardless if 17β-estradiol was added or not (Fig. 6b) . The fusion TF XY1 triggered production of over 120 arbitrary U/g of cellulolytic activity in the same experimental setup [59]. On glucose and glycerol, the expression of cellulases was slightly induced by SynX in the presence of 300 nM 17β-estradiol resulting in approx. 1.7 and 1 arbitrary U/g, respectively (Fig. 6b) . For comparison, the fusion TF XY1 resulted in approx. 10 and 40 arbitrary U/g in the same experimental setup [59]. Next, we wanted to get a more detailed insight into the transactivation activity of SynX and its abilities to induce gene expression of the Xyr1 regulon. To this end, strain TXYE was pre-grown on Mandels-Andreotti (MA) medium [63] containing glycerol as the carbon source without estradiol, because this condition represents a non-induced, non-repressed state. After 24 h of precultivation, equal amounts of mycelium were transferred to MA media containing glucose, glycerol, xylose (mimics induction on xylan [64] ), or lactose, and MA medium without carbon source as a control. The MA media were supplemented with 300 nM 17β-estradiol or only with the solvent DMSO as a control. Samples were taken after 3 and 6 h of cultivation and the total RNA was extracted.

A reverse transcription quantitative PCR (RT-qPCR) assay was performed to measure relative transcript levels of the main Xyr1 target genes. SynX was able to induce the gene expression of the two main xylanases (xyn1 and xyn2) and the aldose reductase xyl1 in dependency of 17β-estradiol on all tested carbon sources (Fig. 7 ). For all three tested genes no or only very low transcript levels were detected when supplemented with DMSO only (Fig. 7) .

The induction of gene expression of the main cellulases, cbh1, cbh2 and egl1 by SynX painted a different picture. We measured high transcript levels of cbh1 on glucose and glycerol in the presence of 17β-estradiol after 3 and 6 h (Fig. 7) . On lactose moderately elevated cbh1 transcript levels were measured in the presence of 17β-estradiol. Without carbon source and on xylose, no induction could be achieved by the addition of 17β-estradiol (Fig. 7) . The transcript levels of cbh2 were distinctively elevated on glucose in the presence of 17β-estradiol. The other measured cbh2 transcript levels are within a single log-unit (Fig. 7) . The same is true for all measured transcript levels of egl1. Notably, the obtained Ct values were all close to the limit of detection. SynX did not induce gene expression of egl1, regardless of carbon source or the presence of 17β-estradiol (Fig. 7) . These results match the previously measured enzymatic activities to the greatest part, i.e. substantial xylanolytic and only low cellulolytic activities (Fig. 6 ).

Previously, the complete hERα was used in filamentous fungi (Aspergillus spp.) [1] , and the LBD of hERα was used in plants in combination with the TAD of VP16 [62] . In this study, we used a synthetic biology approach to evaluate the potential of the LBD of hERα to be used in combination with a TAD from an endogenous TF (i.e. Ypr1) for inducible gene expression in filamentous fungi. We demonstrated earlier that the TAD of Ypr1 can be used to induce the gene expression of Xyr1 target genes nearly carbon source independently by fusing it to the DBD of Xyr1, resulting in the fusion TF XY1 [59]. Here, we further added the LBD of the hERα, resulting in the synthetic TF SynX, with the aim to gain control over the induction process.

Using enzyme activity and RT-qPCR assays, we demonstrated that SynX induces the gene expression of xylanases and the aldose reductase Xyl1 encoding genes in dependency of its inducer 17β-estradiol on any tested carbon source. The induction regulatory system seems to be tight, as we could not detect transcripts or enzyme activities in the absence of 17β-estradiol.

On the other hand, SynX was not able to induce the expression of cellulases to extends worth mentioning. Previously, an overexpression of Xyr1 resulted in a complete abolishment of cellulase production on lactose and elevated cellulolytic activity formation on xylan, whereas the overexpression of the fusion TF XY1 led to elevated cellulolytic activities on lactose and lower levels on xylan than in the Xyr1-overexpression strain [59] . Compared to the enzyme activities in that previous study, we detected moderate cellulolytic activity on xylan and only very low cellulolytic activity on lactose regardless of the presence of 17β-estradiol in TXYE (Fig. 6b) in this study. On glucose and glycerol, the addition of 17β-estradiol resulted in relatively low cellulolytic activities compared to xylan (Fig. 6b) . Thus, the 17β-estradiol-independent cellulolytic activities on xylan and lactose could simply be the result of release from CCR maybe in combination with chromatin opening and the action of other transcription factors. This again demonstrates that the regulation of cellulase expression is a highly complex topic, which still offers surprises and future research possibilities.

The measured cbh1, cbh2, and egl1 transcript levels only partly matched the low cellulolytic activities. We detected virtually no egl1 transcripts on all carbon sources, regardless of the usage of estradiol. Induction of the exo-cellulase encoding genes cbh1 and cbh2 was observed on glucose and glycerol upon addition of estradiol (Fig. 7) . Notably, this induction was a lot less pronounced compared to the one obtained by using TF XY1 in our previous study [59] . The replacement on xylose did not induce genes expression of the cellulase encoding genes compared to a replacement to no carbon source (Fig. 7) , but we could measure enzyme activity on xylan (Fig. 6b) . We reason that the induction experiment with xylose does not simulate the cultivation on xylan entirely.

When we compare the promoter architecture of xylanases and cellulases, we can observe a striking difference. In the xylanase promoters, the Xyr1-binding sites are positioned as inverted repeats, as expected for Gal4-like TFs [65] . In the cellulase promoters on the other hand, the Xyr1-binding sites are arranged as direct repeats [65] . Upon the insertion of an inverted repeat of Xyr1-binding sites into the cbh1 promoter, the CBHI expression was induced on xylan [65] . These results might indicate that Xyr1 has two different modes of action. We suggest the following hypothesis: Xyr1 forms homodimers to bind to the inverted repeats in the xylanase promoters and binds the tandem repeats in the cellulase promoters in a different way. Notably, the native hERα is binding to palindromic sequences (can be viewed as inverted repeats) upon the formation of homodimers [6, 66] . The presence of the LBD of hERα in SynX enforces the formation of homodimers that preferably induce the expression of xylanases but not cellulases.

The measured xylanolytic activities were on average lower in the SynX-bearing strain TXYE than in the before mentioned XY1-bearing strain TXY(1) [59]. For example, TXY(1) reached xylanolytic activities of over 10,000 U/g on glycerol [59], whereas TXYE yielded only approx. 950 U/g in the presence of 300 nM 17β-estradiol (Fig. 6a) . Notably, the used 17β-estradiol concentration (300 nM) was not the optimal concentration for induction of the xylanases on glycerol (Fig. 5b) . On xylan on the other hand, TXYE secreted higher amounts of xylanases (approx. 3700 U/g, Fig. 6a ) than TXY(1) (approx.

These results may indicate a generally lower transactivation capability of SynX than XY1. A simple explanation for this might be unwanted changes of the secondary and/or tertiary structure by intra-molecular interactions of the three domains in SynX. Alternatively, it can be assumed that the LBD of hERα might interfere with protein-protein-interactions between the TAD of Ypr1 and the transcription machinery and/or additional activating factors. Further, we have to consider the influence of other regulatory factors and mechanisms that are responding exclusively to the carbon source, but not to Fig. 7 Transcript levels of the main cellulases-, xylanases-and the aldose reductase encoding genes. The SynX-bearing strain TXYE was pre-cultivated on in shake flasks on glycerol without 17β-estradiol for 24 h. Acquired mycelium was filtered and washed and then equal amounts were transferred to medium containing no carbon source (NC), glucose (Glu), glycerol (Gly), 1 mM xylose (XO), or lactose (Lac) and either 300 nM 17β-estradiol (Estr.) or only the solvent DMSO as control. Samples were taken after 3 and 6 h of incubation, the total RNA was isolated and the relative transcript levels of the indicated genes were determined by a RT-qPCR assay normalized to the reference sample (indicated by an asterisk) using the reference genes sar1 and act1 for normalization. X represents no detection. Error bars indicate standard deviation from three independently grown cultures 17β-estradiol. Notably, not only Xyr1 but also other regulatory factors and mechanism play important roles in the regulation of expression of xylanases and cellulase encoding genes, i.e. the transcription factors Cre1 [38, 39, 67] , Ace1 [46] , Ace2 [47] , Ace3 [43] , Xpp1 [48] and Rce1 [49] , the mating type locus protein Mat1-2-1 [50] , the photoreceptor Env1 [51] , the protein methyltransferase Lae1 [52] , the velvet complex protein Vel1 [53] , the Hap2/3/5 complex [54] [55] [56] , and the MAP kinases Tmk2

[57] and Tmk3 [58] . Further, we observed in a previous study that the DNA packaging adds an important layer to the regulation of expression of xylanase encoding genes [68] . It seems as if T. reesei is integrating a lot of different stimuli using this many different regulators. At the moment, we can only speculate which regulatory factors and/or mechanisms are mediating the carbon source signal (xylan vs. glycerol) and interfere with the induction of cellulase expression by SynX.

In this study we constructed and expressed the synthetic TF SynX (consisting of the DBD of Xyr1, the TAD of Ypr1 and the LBD of the hERα) in T. reesei. SynX successfully induced gene expression of xylanases upon induction with 17β-estradiol and did not activate gene expression without 17β-estradiol. This demonstrates that the LBD of the hERα can be used as inducible regulatory domain in T. reesei. Further, we could demonstrate that the LBD of the hERα is also functional when fused to a TAD other than the commonly used TAD of VP16.

All T. reesei strains (Table 1) For cultivations in shake flasks, T. reesei was grown in 50 ml MA medium containing 1% (w/v) glucose monohydrate, glycerol, xylan from beechwood (Carl Roth), or lactose at 30 °C on a rotary shaker at 180 rpm. A total of 10 9 conidia per liter (final concentration) was used as the inoculum. Mycelia and supernatants were separated by filtration through Miracloth (EMD Millipore, part of Merck KGaA, Darmstadt, Germany). Mycelia were dried at 80 °C over night for biomass determination and supernatants were stored at − 20 °C.

For the replacement experiment, T. reesei was pregrown in 200 ml MA medium containing 1% glycerol at 30 °C on a rotary shaker at 180 rpm. A total of 10 9 conidia per liter (final concentration) was used as the inoculum. Pre-grown mycelia were washed with sterile tap-water, and equal amounts (approx. 0.5 cm 3 ) were resuspended in 20 ml MA medium containing the indicated additives and cultivated at 30 °C on a rotary shaker at 180 rpm.

QM6a Δtmus53 wild-type-like strain with deficiency of the non-homologous end joining repair pathway [73] Xyr1′(81) Xyr1-deficient strain due to a non-sense mutation at position 81 of Xyr1; pyr4 deleted background, no xylanolytic nor cellulolytic activity 

PCRs for cloning purposes were performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs (NEB), Ipswich, MA, USA) according to the manufacturer's instructions. All used primers are listed in Table 2 . PCR products were cloned into EcoRV-digested pJET1.2 (Thermo Scientific, part of Thermo Fisher Scientific Inc., Waltham, MA, USA) and after verification of the PCR products by sequencing (Microsynth, Balgach, Switzerland), they were released for subsequent cloning purposes by digestion with suitable restriction endonucleases (NEB).

For the construction of pRP4-SynX, first, the codonoptimized coding sequence of the hERα part (aa 282-595, Additional file 1, gene synthesis was performed by BioCat GmbH, Heidelberg, Germany) was inserted into the plasmid pJET-Ptef-xyr1N [59] via digestion with MfeI and NheI. Next, the coding sequence for the C-terminal part of Ypr1 was amplified by PCR using the primer Ypr1_L185f-VspI and Ypr1_G674r-MfeI and as template cDNA of T. reesei Δtmus53 grown on glucose, and then inserted into the latter plasmid via digestion with MfeI and NheI. The Ptef::xyr1::hER::ypr1 fragment was released from the resulting plasmid by digestion with Kpn2I and SpeI and inserted into the accordingly digested pCD-RPyr4T [69] .

The protoplast generation and transformation of T. reesei was performed as described previously [70] . Typically, 10 µg of linearized plasmid DNA (in 15 µL sterile ddH 2 O) was used for the transformation of 10 7 protoplasts (in 200 µL). Selection was performed as described previously [69] . Resulting candidates were subjected to homokaryon purification by streaking conidia on plates with selection medium.

Chromosomal DNA was isolated from mycelium by grinding in liquid nitrogen followed by a phenol/chloroform extraction [70] . RNA was degraded using RNaseA (Thermo Scientific). DNA was precipitated with isopropanol, washed with 70% ethanol, and dissolved in ddH 2 O.

For testing the genotype, 10 ng of chromosomal DNA were used as template in a 25-µL-PCR using OneTaq polymerase (NEB) according to the manufacturer's instructions. All used primers are listed in Table 2 . For the agarose gel electrophoresis of the amplification products the GeneRuler 1 kb DNA Ladder was applied (Thermo Scientific).

15 µg of chromosomal DNA were digested with 30 U SpeI (NEB). The resulting DNA fragments were separated by electrophoresis on an 0.8% agarose gel using the GeneRuler 1 kb DNA Ladder for size estimation, then denatured in 0.4 M NaOH, and transferred by capillary forces onto a Biodyne B 0.45 µm nylon membrane (Pall Corporation, Port Washington, NY, USA) using 10 × SSC. 1.5 µg of biotinylated DNA probe were used for hybridization at 65 °C overnight. The probe was generated by PCR with the primers 5pyr4_fwd(BglII) and 5pyr4_rev-BspEI using chromosomal DNA of T. reesei Δtmus53 as template. Labeling of the probe was performed by using a Klenow Fragment (exo-) (Thermo Scientific), random hexamer primers, and biotin-11-dUTP (Jena Bioscience, Jena, Germany). Signals were visualized by using Poly-HRP conjugated to streptavidin and ECL Plus Western Blotting substrate (both Thermo Scientific) on a ChemiDoc MP (Bio-Rad Laboratories, Hercules, USA).

Endo-xylanolytic activities of cultivation supernatants were measured with Azo-Xylan (Megazyme International Ireland, Wicklow, Ireland) according to the manufacturer's instructions. One unit of activity is defined as the amount of enzyme required to release one μmol of reducing-sugar-equivalents per minute. Total cellulolytic enzyme activity of cultivation supernatants were measured using the Cellulase Activity Assay kit (Fluorometric) (abcam189817, Abcam PLC, Cambridge, UK) according to the manufacturer's instructions, with the following adaptions: fluorescence was measured on a Promega GloMax Multi Detection system using the green filter cube (Ex: 520 nm, Em: 580-640 nm), measured fluorescence change rate (Δfluo/min) was used to calculate arbitrary units/ml by multiplying Δfluo/min with 5.1136*10 −4 . Measurements were performed in technical duplicates.

0.01-0.03 g of harvested mycelia were homogenized in 1 mL of peqGOLD TriFast DNA/RNA/protein purification system reagent (VWR, part of Avantor Performance Materials, LLC, Radnor, PA, USA) 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 ONE (Thermo Scientific).

1 µg of isolated RNA were subjected to a DNaseI treatment (Thermo Scientific) according to the manufacturer's instructions and then reverse transcribed using the LunaScript RT SuperMix (NEB) also according to the manufacturer's instructions. The cDNA was diluted 1:50 and 2 µL were used as template in a 15 µL reaction using the Luna Universal qPCR Master Mix (NEB) according to the manufacturer's instructions. All reactions were performed in triplicates on a Rotor-Gene Q system (Qiagen, Hilden, Germany). Calculations of the relative transcript levels were performed according to the Double Delta Ct method [71] using the reference genes sar1 and act1 for normalization according to [72] .

Supplementary information accompanies this paper at https ://doi. org/10.1186/s4069 4-020-00102 -3.

Additional file 1. Partial coding region of the hERα (aa 282-595), codonoptimized for T. reesei, including recognition sites for restriction endonucleases for cloning purposes. 

Metabolically independent and accurately adjustable Aspergillus sp. expression system

Functional domains of the human estrogen receptor

Structural and functional domains of the estrogen receptor

The human estrogen receptor has two independent nonacidic transcriptional activation functions

Characterization of the aminoterminal transcriptional activation function of the human estrogen receptor in animal and yeast cells

Molecular basis of agonism and antagonism in the oestrogen receptor

The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen

The contribution of the N-and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell-specific

Distinct classes of transcriptional activating domains function by different mechanisms

Isoform-selective interactions between estrogen receptors and steroid receptor coactivators promoted by estradiol and ErbB-2 signaling in living cells

The carboxyterminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists

estrogen receptor alpha that are important for modulating transactivation and protein-protein interactions

Molecular determinants of the estrogen receptor-coactivator interface

Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

Molecular mechanism of estrogen-estrogen receptor signaling

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters

A chimeric transactivator allows tetracycline-responsive gene expression in whole plants

Regulation by tetracycline of gene expression in Saccharomyces cerevisiae

A controllable gene-expression system for the pathogenic fungus Candida glabrata

Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans

Adaptable doxycycline-regulated gene expression systems for Drosophila

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

Transcriptional activation by tetracyclines in mammalian cells

Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger

Tet-on, or Tet-off, that is the question: advanced conditional gene expression in Aspergillus

ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger

Establishment of the inducible tet-on system for the activation of the silent trichosetin gene cluster in Fusarium fujikuroi

A light-switchable bidirectional expression system in filamentous fungus Trichoderma reesei

Copper-mediated on-off control of gene expression in filamentous fungus Trichoderma reesei

A universal gene expression system for fungi

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and d-xylose metabolism in Hypocrea jecorina

Chapter 2-Cellulases

The two major xylanases from Trichoderma Reesei: characterization of both enzymes and genes

The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D-galactose catabolism and necessary for beta-galactosidase and cellulase induction by lactose

Xyr1 receives the lactose induction signal and regulates lactose metabolism in Hypocrea jecorina

Regulation of transcription of cellulases-and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form

Cre1, the carbon catabolite repressor protein from Trichoderma reesei

Transcriptional regulation of xyr1, encoding the main regulator of the xylanolytic and cellulolytic enzyme system in Hypocrea jecorina

Differential regulation of the cellulase transcription factors XYR1, ACE2, and ACE1 in Trichoderma reesei strains producing high and low levels of cellulase

Mutation of the Xylanase regulator 1 causes a glucose blind hydrolase expressing phenotype in industrially used Trichoderma strains

Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

A novel transcriptional regulator RXE1 modulates the essential transactivator XYR1 and cellulase gene expression in Trichoderma reesei

The transcription factor ACE3 controls cellulase activities and lactose metabolism via two additional regulators in the fungus Trichoderma reesei

ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression

ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei

Xpp1 regulates expression of xylanases but not of cellulases in Trichoderma reesei

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei

The mating type locus protein MAT1-2-1 of Trichoderma reesei interacts with Xyr1 and regulates cellulase gene expression in response to light

Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

The VELVET A orthologue VEL1 of Trichoderma reesei regulates fungal development and is essential for cellulase gene expression

Chapter 34-evaluation of hormonal status

Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants

Applications of cellulases

D-Xylose as a repressor or inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei)

Influence of cis element arrangement on promoter strength in Trichoderma reesei

Estrogen receptor interaction with estrogen response elements

Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei

Impact of xylanase expression-inducing compounds on DNA accessibility in Trichoderma reesei

Novel strategies for genomic manipulation of Trichoderma reesei with the purpose of strain engineering

The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain

A new mathematical model for relative quantification in realtime RT-PCR

An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei)

Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)

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Not applicable.

CD constructed all plasmids and strains, performed the cultivation experiments, the enzyme and RT-qPCR assays, was involved in the conception of the study, and wrote the manuscript. RLM was involved in the conception of the study and revised the manuscript. ARMA supervised the experiments, was involved in the conception of the study, and revised the manuscript. All authors read and approved the final manuscript.

This study was supported by a Grant from the Austrian Science Fund (FWF): P26733-B22 given to ARMA.

All data and materials described are freely available for scientific and academic purposes upon request to the corresponding author.

No human or animal subjects were utilized in the course of this work.

Not applicable.

The authors declare that they have no competing interests.Received: 27 April 2020 Accepted: 17 July 2020