l-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei
© Bischof et al. 2015
Received: 18 June 2015
Accepted: 30 July 2015
Published: 14 August 2015
Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the l-methionine repressible MET3 gene, encoding ATP sulphurylase.
We show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are l-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including d-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei.
We describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an l-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.
Trichoderma reesei is a widely employed microorganism for the production of technical enzymes, including cellulases and hemicellulases that are used by a variety of industries including the biorefinery industry [1–3]. As a result, a relatively well developed toolbox for the genetic engineering of T. reesei has become available over the past years . Tuneable promoters are essential tools in this respect and represent a versatile means to alter gene expression at the level of gene transcription . Repressible promoters in particular are useful because (1) they allow the conditional switch-off of genes when e.g. the permanent deletion of that gene is lethal; and (2) they allow the transient control of metabolic pathways, e.g. to slow down metabolism of an intracellular substance leading to its accumulation. Both strategies are equally important for strain construction of industrial producer strains as well as functional genomics studies. In the context of industrial enzyme production, the use of an inexpensive repressing substance that is effective at low concentrations is mandatory. However, a repressible promoter meeting these specifications is currently not available for T. reesei, where tuneable expression is almost exclusively controlled by the promoters of the cellobiohydrolase cel7a or endoxylanase xyn1 . In these two cases, the repression is mediated by d-glucose [6, 7], which in turn leads not only to the repression of cellulases and xylanases but also to the repression of most of the other carbohydrate active enzymes (CAZYmes) [8, 9] which is usually undesirable. Repressible expression systems that have been proven to work in filamentous fungi and yeast include the thiamine repressible thiA system of Aspergillus fumigatus and Aspergillus oryzae [10, 11] and the copper dependent Saccharomyces cerevisiae CTR4 system in Schizosaccharomyces pombe and Cryptococcus neoformans [12–14]. Both systems have the advantage of responding to µM concentrations of the respective modulating substances, but suffer from the fact that these compounds may already be present in industrially used complex carbon sources such as wheat straw. Another widely employed system for different yeasts including S. cerevisiae, Ashbya gossypii, Candida albicans or Pichia pastoris is the l-methionine repressible MET3 system [15–18], which however has not been tested in filamentous fungi so far. We regard l-methionine as a promising repressor because its addition to T. reesei cultures was previously shown to have no effect on growth and CAZyme expression . Owing to its use as a food and feed additive  it is also modestly prized and abundantly available. However, we show here that the expression of the T. reesei met3 orthologue responds to l-methionine in the same way as the S. cerevisiae gene only when lactose, but not when a complex carbon source such as wheat straw is used as the carbon source. Since wheat straw is one of the most relevant feedstocks for biorefineries [9, 21], we developed a repressible expression system for this carbon source. Using a transcriptomic approach, we identified a novel set of l-methionine repressible genes that feature a diverse range of basal expression strengths and degrees of achievable repression. We also demonstrate the applicability of the promoter region of the most strongly expressed of said genes for tightly repressible expression of intra- and extracellular reporter genes on wheat straw as well as on d-glucose and glycerol containing media.
Repression of the T. reesei met3 (ATP sulphurylase-encoding) gene by l-methionine
Genome-wide identification of l-methionine repressible genes
l-Methionine regulated genes of wheat straw grown T. reesei QM9414 cultures
Log2 gene expression from array data
Taurine catabolism dioxygenase TauD/TfdAa
MSF permease, allantoin-permease likec
Urea transporter/Na+-amino acid transporterc
DszA-type family xenobiotic monooxygenasesa
DszA-type family xenobiotic monooxygenasesa
Amino acid transporter, high affinity methionine transporterc
Taurine catabolism dioxygenase TauD/TfdAa
MSF permease, allantoin permease likec
Urea transporter/Na+ amino acid transporterc
Assimilatory sulfite reductase, alpha subunitb
Taurine catabolism dioxygenase TauD/TfdAa
Unknown protein GPR1/FUN34/yaaH-like
Amino acid transporterc
Phosphoadenosine phosphosulfate reductaseb
Phosphoenolpyruvate carboxykinase AcuF
DszA-type family xenobiotic monooxygenases
Amino acid transporter, AAT-familyc
Sulfat transporter SUL1b
DszA-type family xenobiotic monooxygenasesa
CE5 acetyl xylan esterase
Siderophore biosynthesis lipase/esterasea
NRPS siderophore synthase SID1a
DIP2, encoding a nucleolar protein
C2H2 transcriptional regulator
Short chain dehydrogenase/reductase
Shows similarity to S. cerevisiae Utp11
Unknown protein, GFA-domain
Unknown protein, secreted, only in hypocreaceae, HTG
UTP10, encoding a component of the SSU processome
Construction of T. reesei strains with repressible lacZ and sucA reporters
We then selected the promoter (subsequently termed P 123979 ) of the TauD like dioxygenase-encoding gene trire2:123979 for further testing, because it displayed the highest level of transcription of this gene family in the absence of exogenous l-methionine (log2 fluorescence of 12.75) and strong repression in the presence of 0.1 mM added l-methionine (see Table 1). As reporter genes, we chose a modified version of Aspergillus niger invertase encoding sucA (ß-d-fructofuranosidase, EC 18.104.22.168; ) and the E. coli ß-galactosidase encoding lacZ, respectively. As a comparison to the expression strength of P 123979 , we used the strong constitutive promoter of tef1 (P tef1 ) as previously described [30, 31]. Both promoter regions were inserted into sucA and lacZ expression plasmids that also contained the hphB resistance cassette for selection. After transformation of the sucA expression cassettes into T. reesei QM9414 and purification of the transformants, we isolated at least three positive strains for the repressible promoter and two P tef1 reference strains having a single integration of the construct as verified by qPCR (see Additional file 1: Figure S2). In the same way, we also constructed strains expressing E. coli lacZ under the control of the promoters of P 123979 and P tef1 and again isolated strains having a single copy integration of the expression cassette (see Additional file 1: Figure S3).
l-Methionine dependent repression of sucA expression on wheat straw medium
Portability of the l-methionine repressible expression system to other carbon sources and alternative repressors
Repression of LacZ activity in T. reesei d-glucose and glycerol cultures
P 123979 -lacZ 3
1.27 ± 0.15
0.04 ± 0.01
1.02 ± 0.07
P 123979 -lacZ 7
1.46 ± 0.12
0.05 ± 0.00
0.81 ± 0.31
P 123979 -lacZ 3
1.09 ± 0.08
0.09 ± 0.02
0.03 ± 0.00
P 123979 -lacZ 7
1.02 ± 0.59
0.10 ± 0.00
0.05 ± 0.00
l-cysteine is often used as an alternative repressor for MET3 orthologues in different yeasts and is linked to l-methionine metabolism via homocysteine and cystathionine (Additional file 1: Figure S1). When we tested the effect of l-cysteine on P 123979 repression, only the 5 mM concentration had an equally pronounced effect, whereas concentrations of 1 mM resulted only in a minor reduction of ß-galactosidase activity. Since repression by lower concentrations of l-methionine appeared as a transient phenomenon in this study, we also tested the effect of the non-metabolizable substitute l-ethionine on two of the P 123979 –sucA strains. However, as shown in Fig. 4, l-ethionine addition failed to reproduce the tight repression observed with l-methionine 2 h after its addition and about a third of the initial sucA mRNA could still be detected. After 5 h, the transcript level even surpassed the basal level before l-ethionin addition more than threefold which shows that l-ethionin cannot be used for repression of P 123979 .
l-methionine as a repressor of gene expression in yeasts is well established. In S. cerevisiae, repression of the l-methionine biosynthesis genes MET2, MET3, MET5, MET6, MET14 and MET25 has been shown [17, 32] and the promoters of the MET3 orthologues of C. albicans, A. gossypii and P. pastoris have been used for repressible gene expression [15, 16, 18]. Our results show that when a complex carbon source such as pretreated wheat straw is used, only the phosphoadenylyl-sulfate reductase encoding met16 (trire2:65410), sulfite reductase encoding met5/ecm17 (trire2:81576), homoserine O-acetyltransferase encoding met2 (trire2:66517) and sulfate permease encoding sul1 (trire2:79741) are more than twofold repressed by 0.1 mM l-methionine in T. reesei. In contrast, the orthologue of sulfate adenylyltransferase encoding met3 (trire2:47066) was strongly repressible by l-methionine at concentrations higher than 0.1 mM only when lactose was used as the carbon source and the degree of repression was comparable to that observed in S. cerevisiae [17, 32].
To identify novel repressible promoters we used therefore a genome-wide screen under l-methionine repressing conditions. We found genes for transport proteins to be a highly enriched group in the downregulated transcriptome of T. reesei confronted with l-methionine. Two of the transporters that were downregulated in the presence of l-methionine appear to be high affinity amino acid transporters and one of them indeed shows moderate sequence similarity to the characterized S. cerevisiae l-methionine and l-cysteine transporter Mup1 (, blastp score 4.7 E−78). It is therefore tempting to speculate that the uptake of l-methionine itself represents another control circuit for the maintenance of l-methionine homeostasis in T. reesei. Repression of l-methionine transport by l-methionine at the transcriptional level also occurs in Penicillium chrysogenum and S. cerevisiae [34, 35] as well as in E. coli . Our findings that a third of the genes that were repressed by 0.1 mM l-methionine encoded proteins involved in iron acquisition or contained iron suggested a relationship between iron metabolism and l-methionine availability. Recently, Amich et al.  have demonstrated a cross-talk between iron homeostasis and sulphur metabolism: starving an Aspergillus fumigatus mutant defective in the sulfur metabolite transcriptional repressor MetR for sulphur results in increased expression of the genes of the iron regulon. The molecular basis for this cross-talk is the upregulation of iron acquiring proteins when the biosynthesis of Fe-S cluster enzymes is impaired because of a shortage of l-cysteine . Consequently, loss-of-function of MetR—and thus sulfur derepression—results in increased cellular iron as well as ferricrocin contents accompanied by transcriptional derepression of genes involved in iron acquisition such as siderophore biosynthesis and uptake as well as reductive iron assimilation . Our transcriptomic results under l-methionine addition (=sulfur repression) are essentially the opposite of those with the A. fumigatus MetR mutant and would therefore be in perfect agreement with the operation of an iron-sulfur crosstalk also in T. reesei. However, the strong repression of the iron containing oxygenases may also have an alternative explanation: in bacteria, Fe2+/α-ketoglutarate dependent TauD/TfdA like dioxygenases are involved in the release of sulfur from the aminosulfonic acid taurine and other organic sources of sulfur . In E. coli, their transcription as well as that of other sulfur starvation induced genes is positively influenced by the transcription factor Cb1 [40–42]. A negatively regulating element in this system is the sulfur assimilation metabolite adenosine 5′-phosphosulfonate, which binds Cb1 and abolishes its activating function [43, 44]. Similarly, dibenzothiophene sulfone monooxygenase (DszA) is involved in biodesulfurization in Rhodococcus erythropolis [45, 46] and repressed by both l-methionine and l-cysteine. If a similar regulation occurs in fungi, it would offer an alternative explanation for the negative regulation of these oxygenases by l-methionine. This in turn would mean that this system can likely also be transferred to other species, because TauD/TfdA like dioxygenases are in fact quite common in other Pezizomycotina and particularly also among industrially relevant Aspergillus spp. and the plant pathogenic Fusarium spp. (Additional file 1: Figure S4). Although the transient nature of l-methionine repression of the P 123979 promoter at low concentrations is advantageous to effectively deactivate and reactivate gene expression, it might also be useful to have a repression system that is tightly shut by low concentrations of a repressor during a longer period of time. Attempts to use the l-methionine non-metabolizable analogue l-ethionine for this purpose failed, which probably points to the fact that—like in S. cerevisiae and A. nidulans [47, 48]—S-adenosylmethionine, rather than l-methionine itself, is the repressing metabolite. l-Ethionine cannot be adenylated and inhibits the SAM-dependent methyltransferases . An alternative to overcome this bottleneck would be to knock out the gene further metabolizing S-adenosylmethionine (i.e. adenosyl-homocysteine hydrolase) or placing it under the control of an l-methionine repressible promoter.
In the present manuscript we describe novel l-methionine repressible genes that serve as an alternative to the well-known S. cerevisiae MET3 system under conditions where its T. reesei orthologue met3 is non-repressible, i.e. when the industrially relevant carbon source wheat straw is used. The described set of repressible genes covers a wide array of expression strengths and levels of achievable repression. We demonstrate that we can use the promoter region of a TauD/TfdA-like dioxygenase to achieve repressible gene expression of two reporter genes in T. reesei and that this system can also be used on less complex carbon sources such as d-glucose or glycerol and is likely transferable also to other fungi. Taken together these findings show that the present system offers a versatile means to realize repressible gene expression and therefore represents a valuable tool for industrial strain engineering and functional genetics of T. reesei.
Strains and culture conditions
Trichoderma reesei QM9414 (ATCC 26921) was used throughout the study and maintained on potato dextrose agar (Difco). Cultivation of T. reesei with pretreated wheat straw, d-glucose, glycerol or lactose as the sole carbon source was routinely performed as previously reported . In brief, pretreated wheat straw up to a final concentration of 10 g L−1 dry weight was added to unbuffered mineral medium, the pH set to 4.8 with 1 M KOH and then autoclaved. CaCl2 was added separately after autoclaving as were the other carbon sources. 1 × 106 spores ml−1 were then aseptically added to 200 ml of the medium in a 1 L Erlenmeyer flask. The culturing conditions were 28 °C and 250 rpm in a rotary shaker. For the cultivations where extracellular invertase activity was measured, a slightly modified medium that contained per liter: 5 g (NH4)2SO4, 2 g KH2PO4, 0.3 g MgSO4, 0.4 g CaCl2, 1.7 g yeast nitrogen base without amino acids and (NH4)2SO4, 10.22 g (i.e. 50 mM) potassium phthalate, 20 g pretreated wheat straw, 5 mg FeSO4 × 7H2O, 1.6 mg MnSO4 × H2O, 1.4 mg ZnSO4 × 7 H2O, 1.61 mg CoCl2 and 0.1 mg CuSO4 × 5 H2O was used.
Trichoderma reesei QM9414 cultures were pregrown for 48 h on wheat straw medium after which 0.1 mM of l-methionine were added to the cultures. Mycelia were harvested after 2.5 h, washed and instantly frozen in liquid nitrogen. Genome wide gene expression analysis was performed as previously described . The microarray data and the related protocols are available at the GEO web site (http://www.ncbi.nlm.nih.gov/geo/) under accession number: GSE69745. Groups of eukaryotic orthologous genes (KOG) were retrieved from the T. reesei genome website (http://genome.jgi-psf.org/cgi-bin/kogBrowser?db=Trire2). When no KOG assignment was available there, sequences were manually assigned after sequence analysis using Interpro (http://www.ebi.ac.uk/interpro/) and OrthoDB (http://orthodb.org/orthodb7). In ambiguous cases, KOG category S for “unknown function” was assigned. Enrichment of KOG groups was determined by Fisher’s exact test.
Analysis of the l-methionine biosynthesis pathway and related pathways in T. reesei
Protein sequences were retrieved from the KEGG catalogue of the JGI T. reesei portal (http://genome.jgi-psf.org/cgi-bin/metapathways?db=Trire2) and verified by comparison to the NCBI database using blastp.
Reverse transcription qPCR
Culture conditions were identical to the cultures from the transcriptome analysis if not stated otherwise and methods for total RNA extraction and qPCR measurements are described in detail elsewhere . Primers sequences and amplification efficiencies are given in the supplementary material (Additional file 1: Table S2). Determination of the PCR efficiency was performed using triplicate reactions from a decadic dilution series of cDNA, and the amplification efficiency then calculated from the given slopes using the realplex v2.2 software. Expression ratios were calculated using REST© software .
Construction of sucA and lacZ reporter vectors
The sucA reporter gene was de novo synthesized by MWG Operon (Ebersberg, Germany), whereby a 6x His-tag was introduced C-terminally and the native SucA signal peptide was replaced by the signal peptide from T. reesei CEL7A (MYRKLAVISAFLATARA). Also, two restriction sites (SalI and ClaI) of the sucA ORF were removed by silent mutations (Codon526:CAC⇒GAC; Codon541:AAT⇒GAT). Constructions were made in pBS_gfp_ThH3_hph, an egfp expression vector consisting of the egfp gene, a 369 bp terminator fragment of the histone 3 (hH3) terminator and the hph expression cassette  as selection marker (Robert Bischof and Bernhard Seiboth, unpublished data). The sucA gene was inserted into the ClaI/SalI fragment of pBS_gfp_ThH3_hph replacing egfp and resulting in pRB-X. Fragments of the candidate l-methionine repressible promoter P123979 and the tef1 promoter region were amplified from chromosomal DNA of T. reesei QM9414 using primers Inf_pRB-XVI_fw and Inf_pRB-XVI_rv and Inf_pRB-XVIII_fw and Inf_pRB-XVIII_rv, respectively and subsequently introduced into the SalI site of pRB-X by recombinational cloning with the Infusion®HD Cloning Kit (ClonTech, CA, USA) resulting in plasmids pRB-XVI (P 123979 -sucA) and pRB-XVIII (P tef1 -sucA), respectively. Similarly, the lacZ gene (GenBank:NC_000913) was de novo synthesized, the ClaI site removed (Codon 280 ATC⇒ATT) and inserted into the SalI/ClaI site of pBS_gfp_ThH3_hph resulting in pRB-XI. For the construction of lacZ reporter vectors, the lacZ β-complementation region of pLHhph  was eliminated by PCR amplification of the vector using primers hphF and pLHhph-lacZ_rv. The PCR mix was then digested with DpnI to remove the template DNA by methylation specific restriction. The promoter fragments of trire2:123979 (i.e. P 123979 ) and tef1 (P tef1 ) were amplified from chromosomal T. reesei DNA using primers XVII_inf_P_fw and XVII_inf_P_rv and XIX_inf_P_fw and XIX_inf_P_rv, respectively. The lacZ gene was amplified from pRB-XI using primers XVII_inf_L_fw and XVII_inf_L_rv (for P 123979 ) and XIX_inf_L_fw and XIX_inf_L_rv (for P 123979 ), respectively. Both fragments were simultaneously inserted into the pLHhph-lacZ DNA fragment via Infusion®HD Cloning Kit resulting in pJH-III (P 123979 -lacZ) and pJH-IV (P tef1 -lacZ). Oligonucleotides are given in the supplementary material (Additional file 1: Table S2).
Fungal transformation and strain characterization
Recombinant T. reesei strains were generated by protoplast mediated transformation as described earlier  using MEX plates containing 100 µg ml−1 hygromycin for selection. Transformants were then passaged to PDA plates containing the same amount of hygromycin and subsequently purified twice via single spore isolation on PDA plates containing 0.1 % (w/v) Triton X-100 as colony restrictor. DNA extraction, PCR screening and gene copy number estimation by qPCR was done essentially as described by Tisch et al. . Primer sequences for the PCR screening are given in the supplementary material (Additional file 1: Table S2) and positive (plasmid DNA) as well as negative controls (genomic DNA of T. reesei QM9414) were included in the PCR screening.
Measurement of extracellular invertase activity
5 ml culture broth samples were prefiltered through Miracloth tissue and centrifuged. Supernatants were immediately snap-frozen in liquid nitrogen and kept on −20 °C until they were analyzed. Invertase activity towards 12.5 mM saccharose in the supernatant was assayed in 50 mM sodium citrate buffer (pH = 4.6) at 25 °C for 20 min and the reaction stopped by the addition of 50 mM Trizma base. Each sample was assayed as a technical triplicate. The released d-glucose was assayed using a commercially available d-glucose assay reagent (Sigma, St. Louis, MO, USA). Protein concentrations were determined with the Bio-Rad protein assay using BSA as standard (Biorad, Hercules, CA, USA). Non inoculated substrate controls were included in both cases and the absorbances read out using a Spectra Max Plus 384 microplate reader (Molecular Devices, Sunny Vale, CA, USA). Coefficients of variation were typically at or below 5 %. One unit was defined as the amount of enzyme, which hydrolyses 1 µmol of sucrose to d-glucose and d-fructose per minute.
Measurement of intracellular ß-galactosidase activity
The mycelium from 10 ml of culture broth was harvested by filtration with Miracloth tissue, washed with 50 ml ice cold tap water and snap-frozen in liquid nitrogen. Mycelia were stored at −80 °C until analyzed. Cell extracts were prepared as principally described elsewhere  but using EDTA free cOmplete ULTRA Tablets (Roche, Dubai, UAE) instead of phenylmethylsulfonfluorid and pepstatin A only. ß-galactosidase activity towards 20 mM ortho-nitrophenyl-ß-d-galactosid (ONPG) was assayed in 10 mM phosphate buffer (pH = 7.4) at 40 °C for 20 min. Each sample was assayed as a biological and technical triplicate. The nitrophenol content of the hydrolysis reaction was directly read out using a Spectra Max Plus 384 microplate reader and the activity calculated using Lambert–Beer’s law and a molar extinction coefficient of 18.5 ml mol−1 cm−1 for nitrophenol. The intracellular protein content was assayed as described above. One unit was defined as the amount of enzyme, which hydrolyses 1 µmol of ONPG to ß-d-galactose and nitrophenol per minute.
RB planned and carried out experiments, analysed and interpreted data and wrote the manuscript; JH carried out experiments, analysed and interpreted data; BM planned and carried out experiments, analysed and interpreted data; CG initiated the study, interpreted and discussed experimental data and was involved in the design of the study; BS and CPK initiated and designed the study, planned experiments, interpreted and discussed data and edited the manuscript. All authors read and approved the final manuscript.
This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and ZIT—Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. We also want to thank Claudia Koger, Alexander Beinhauer and Teresa Deckardt for their technical assistance.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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