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Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei

Abstract

Background

The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.

Results

In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.

Conclusion

With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.

Background

The filamentous ascomycete Trichoderma reesei (teleomorph Hypocrea jecorina [1]) is used for the industrial production of cellulases and xylanases and has established itself as model organism for several aspects of fungal biology including regulation of gene expression, protein secretion, sexual development, and light response [2,3,4,5,6,7]. Trichoderma reesei has been in the focus of basic and applied research for several decades [8, 9] and holds a great promise for heterologous protein expression and secretion due to its outstandingly high protein secretion rate [2, 5]. A fundamental prerequisite for controlled heterologous protein expression is the ability to insert genes at defined loci. In a previous study, we developed a strategy for targeted gene insertions using auxotrophic markers in T. reesei [10]. In that study, we demonstrate that the upstream regions of the pyr4 gene [encoding for the orotidine 5′-phosphate decarboxylase (EC 4.1.1.23)] and the asl1 gene [encoding for the argininosuccinate lyase (EC 4.3.2.1)] as target sites for gene insertions. In a first step the promoters and the complete or partial coding regions of the genes are deleted, leading to uridine and arginine auxotrophy, respectively. The resulting strains can be used as recipient strains for gene integrations; a gene of interest is inserted upstream of the promoter regions together with the previously deleted genomic sequences. Please refer to our previous study for a detailed description of this strategy [10]. This yields strains that are isogenic to the original parent strain except for the inserted gene. Prototrophy is simultaneously re-established and can be used for selection of the gene insertion.

In this study, we describe the applicability of the his1 gene [TRIREDRAFT_80820, encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17)] as a suitable insertion locus and auxotrophic marker for gene integrations in T. reesei. Additionally, we test if and how the choice of the integration site effects the expression of the inserted gene. To this end, we determine the expression of the reporter EYFP (enhanced yellow fluorescence protein) in strains carrying the eyfp gene at the pyr4, the asl1, or the his1 locus by comparative transcript analysis and fluorescence measurements. Additionally, we describe a minimal effort and seamless marker recycling strategy, and we construct a triple auxotrophic strain, which can be used for future multiple gene insertions.

Results

Deletion of his1 leads to histidine auxotrophy in T. reesei

First, we deleted a part of the his1 coding region and the native promoter using a homologous recombination strategy and the pyrG marker (from Aspergillus fumigatus) (Fig. 1A). To this end, the plasmid pCD-Δhis1 was linearized and transformed into T. reesei QM6a Δpyr4. The correct integration was verified by PCR analyses (Additional file 1: Figure S1). The resulting strain, T. reesei QM6a Δhis1(pyrG  +) was histidine auxotroph and prototroph for uridine because the A. fumigatus pyrG complemented the pyr4 deletion (Fig. 2). Notably, the deletion of his1 also lead to a reduced growth rate on supplemented minimal and malt extract medium, delayed the onset of conidiation, and reduced the total amount of spores (not shown).

Fig. 1
figure1

Modification of the his1 locus during strain generations. A In the uridine auxotrophic recipient strain T. reesei QM6a Δpyr4, the his1 gene (blue arrow) is located in close vicinity to two other genes (grey arrows; TRIREDRAFT_67534 is a predicted protein kinase, TRIREDRAFT_23028 is a hypothetical Ca2  +  permeable channel). After transformation of the plasmid pCD-Δhis1, homologous recombination may occur at the two flanks (orange and yellow boxes) resulting in the replacement of the his1 promoter and a part of the coding sequence with the A. fumigatus pyrG marker (green arrow), which restores uridine prototrophy. This yields the strain T. reesei QM6a Δhis1 (pyrG  +). B Due to the direct repeat of a part of the 5′flank (dark orange box) in front of the 3′flank (yellow box) in the strain T. reesei QM6a Δhis1 (pyrG  +) an internal homologous recombination may occur spontaneously, which leads to the loss of the pyrG gene. This results in uridine auxotrophy and the generation of the double-auxotrophic strain T. reesei QM6a Δpyr4 Δhis1. C Transformation of the plasmid pCD-ReHis-eyfp into the strain T. reesei QM6a Δpyr4 Δhis1 may lead to a homologous recombination at the 5′ and 3′flanks (orange and yellow boxes). As the plasmid contains the previously deleted his1 promoter and partial coding region, the native his1 locus is restored and additionally an EYFP expression cassette integrated upstream of the his1 promoter, yielding the strain QM6a Δpyr4 eyfp(his1)

Fig. 2
figure2

Phenotype characterization of the auxotrophic strains. The indicated strains were cultivated on minimal medium plates supplemented with 5 mM uridine, 2.5 mM arginine, and 4 mM histidine (MM  +), and on comparable plates lacking one of the three supplements (-Uri, -His, -Arg), and on minimal plates without any supplements (MM-)

The deletion cassette contained a partial, direct repeat of the 5′flank in front of the 3′flank (Fig. 1A). The duplication of this approx. 400 bp long sequence may lead to an internal homologous recombination event, which results in a loss of the previously integrated pyrG gene (Fig. 1B). This event may occur randomly without an external stimulus. We selected for cells in which this internal homologous recombination event happened by cultivation the T. reesei QM6a Δhis1(pyrG  +) strain on a plate containing 5-FOA (Additional File 2). This strategy enables seamless marker recycling because no genetic traces of the initially integrated pyrG remain at the locus (Fig. 1B). The loss of the pyrG gene was verified by a PCR assay (Additional File 1: Figure S1). As expected, the resulting strain, T. reesei QM6a Δpyr4 Δhis1 was auxotrophic for histidine and uridine (Fig. 2) and grew slower and exhibited delayed and reduced conidiation compared to the T. reesei QM6a Δpyr4 (not shown).

Targeted gene insertion at the his1 locus

Next, we tested, whether we can use the his1 locus as insertion site for a targeted gene integration, and whether we could use the his1 genes as selection marker for the transformation. To this end, we transformed the linearized plasmid pCD-ReHis1-eyfp into T. reesei QM6a Δpyr4 Δhis1 (Fig. 1C) and selected for the reestablishment of histidine prototrophy. The correct integration was verified by PCR analyses (Additional file 1: Figure S2). The resulting strain T. reesei QM6a Δpyr4 eyfp(his1) was still auxotrophic for uridine but had regained prototrophy for histidine (Fig. 2) and was expressing EYFP (Fig. 3). The reinsertion of his1 also restored normal growth rate and conidiation behavior like in the parent strain T. reesei QM6a Δpyr4 on minimal and malt extract medium (not shown).

Fig. 3
figure3

Expression analysis of EYFP on transcript and enzyme level. A The EYFP reporter strains (Table 1) carrying the expression cassette at either the pyr4, the asl1, or the his1 locus were cultivated in a 12-well plate in 1.5 ml MAM containing glucose, lactose, glycerol, or xylan as carbon source. After incubation at 30 °C for 48 h, RNA was extracted, and cDNA was synthesized. The relative transcript levels of the eyfp were determined in a RT-qPCR assay using act1 and sar1 for normalization and the Pfaffl method [15] for calculation. QM6a eyfp(pyr4) on glucose was used as reference sample. The arithmetic average of all samples from all carbon sources are depicted in the bar chart. Error bars represent standard deviation. B The strains T. reesei QM6a Δpyr4, and the EYFP reporter strains carrying the expression cassette at either the pyr4, the asl1, or the his1 locus were cultivated in a fluorescence 96-well plate in MAM containing glucose, lactose, glycerol, or xylan as carbon sources. After incubation at 30 °C without agitation for 72 h, the total fluorescence (ex 490, em 510–570) was measured. Bars represent the arithmetic average of three independent replicates. Error bars represent standard deviation

Effects of integration site on gene expression

In a previous study, we constructed two other EYFP expression strains analogously to T. reesei QM6a Δpyr4 eyfp(his1), namely QM6a eyfp(pyr4) and QM6a eyfp(asl1). These three strains carry the very same eyfp expression cassette at the his1, the pyr4 and the asl1 locus, respectively [10]. Notably, each strain bears only a single copy of the eyfp gene (Additional File 3; [10]). Next, we tested if and how of the insertion locus effects the gene expression of the EYFP marker. To this end, we cultivated the three latter strains together with T. reesei QM6a Δpyr4 on different carbon sources and measured the transcript levels of eyfp (Fig. 3A) and the EYFP fluorescence (Fig. 3B). As the experimental setup does not allow determining the biomass, the fluorescence units could not be normalized to the acquired biomass. However, the strains grew equally fast on the used carbon sources in a parallel cultivation in clear well plates (Additional File 4). Consequently, the fluorescence values of the different strains grown on the same carbon sources can be compared. We observed approx. two-fold higher eyfp transcript levels (Fig. 3A) and significantly higher fluorescence (Fig. 3B) in the strain carrying the EYFP expression cassette at the pyr4 locus compared to the other two strains, which were similar to each other (Fig. 3B; Additional File 5). This demonstrates that all three loci can be used for heterologous gene expression and that the choice of the integration locus influences the gene expression.

Construction of a triple auxotrophic recipient strain

Next, we decided to construct a recipient strain for multiple gene insertions for future studies and applications. To this end, we transformed the linearized plasmid pCD-Δasl1 [10] into T. reesei QM6a Δpyr4 Δhis1 and selected for hygromycin resistance, because the deletion cassette contains the corresponding resistance gene (Additional File 1: Figure S3). Please refer also to [10] for a detailed description and depiction of the asl1 deletion strategy. The deletion of the asl1 promoter and part of the coding region, was confirmed by a suitable PCR analysis (Additional File 1: Figure S3). The resulting strain T. reesei QM6a Δpyr4 Δhis1 Δasl1 was auxotrophic for uridine, histidine, and arginine (Fig. 2) and may be used as recipient strain in the future.

Discussion

In this study, we demonstrated that the his1 locus can be used as integration site for gene expression cassettes and that the his1 gene can be used as auxotrophic marker in T. reesei. We observed that the deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy, but also negatively affected the growth rate and conidiation. We speculate that this might be a result of the connection of the histidine and purine biosynthesis pathways [11]. During one reaction of the histidine biosynthesis pathway, AICAR (5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-Monophosphate) is formed as co-product (https://www.genome.jp/entry/R04558). AICAR is an important intermediate for the biosynthesis of purines and is involved in other biological processes [11]. It appears, that AICAR cannot be provided in sufficient amounts through other metabolic pathways in the his1 deletion strains. However, the re-establishment of the his1 locus re-instates prototrophy and normal growth and sporulation behavior. This needs to be considered for the design of a strain construction strategy; the final strain must contain a functional his1 locus.

In a previous study, we described the applicability of two other genes, pyr4 and asl1 for targeted gene insertions [10]. We routinely use the therein described strains and the markers in our research group, because no expensive or toxic antibiotics are needed, and the resulting strain do not carry additional marker genes, which might interfere with the planned gene expression. In this study, we demonstrated that the choice of the integration locus has a strong influence on the gene expression. This should also be considered for the strain design. The combination of differently strong promoters with different integration sites may facilitate fine-tuning of the final gene expression rate. This is of course highly speculative and should be tested in further studies.

When comparing the EYFP expression in the three eyfp bearing strains, we observed a strong influence of the different carbon sources on the fluorescence, but not on transcript levels (small standard deviation in the transcript analysis). This seems contradictory at first glance but can be explained by the different growth rates of T. reesei on the tested carbon sources. We speculate that eyfp is transcribed at a constant rate regardless of the carbon source, but the different growth rates on the different carbon sources lead to different amounts of acquired biomass which in turn produces and accumulates more or less EYFP. A normalization to the biomass would probably solve this problem, but the performed experiment did not allow determining the biomass in the fluorescence well plates.

Further, we described a minimal effort and seamless marker recycling method, that relies on an internal homologous recombination between two direct repeats of a natural genomic sequence (Fig. 1B). This is a random and spontaneous process that may occur during the normal cell cycle of T. reesei. It is also interesting to speculate how and if CRISPR-mediated genome editing may be combined with the here presented minimal effort and seamless marker recycling method. If a suitable recognition site for the Cas9 enzymes is generated by the internal homologous recombination, CRISPR may be used to open the target site for enhanced transformation and integration efficiency.

Conclusions

We could demonstrate the applicability of the his1 gene for targeted gene integration and as an auxotrophic marker in T. reesei, which expands the toolbox for future applications of this fungus as host for heterologous gene expression. We further demonstrated the applicability of a minimal effort and seamless marker recycling system, which will facilitate future strain construction efforts, because several genomic manipulations may be performed without the need of several marker genes and/or expensive and toxic compounds.

Methods

Fungal strains and cultivation conditions

All T. reesei strains (Table 1) used in this study were maintained on malt extract agar at 30 °C. Uridine, Arginine, Histidine, 5-FOA, and Hygromycin B were added when applicable to a final concentration of 5 mM, 2.5 mM, 4 mM, 1 mg/ml, and 113 U/ml, respectively.

Table 1 T. reesei strains used in this study

For cultivations, T. reesei was grown in Mandels-Andreotti medium (MAM) (8.9 g/L Na2HPO4∙2 H2O, 1.4 g/L (NH4)2SO4, 2 g/L KH2PO4, 0.3 g/L MgSO4, 0.4 g/L CaCl2, 0.3 g/L urea, 1 g/L peptone, 20 mL/L trace elements (5 mg/L FeSO4∙7 H2O, 1.6 mg/L MnSO4∙H2O, 1.4 mg/L ZnSO4∙H2O and 2 mg/L CoCl2∙2 H2O), pH adjusted to 5 with citric acid) [12] containing 1% (w/v) of the respective carbon source. Culture were either grown in 20 ml in Erlenmeyer flasks in a rotary shaker at 30 °C and 180 rpm, or in 100 µl in fluorescence 96-well plates (sterile, flat bottom, black) at 30 °C without agitation. A total of 109 conidia per liter (final concentration) was used as the inoculum in both cases.

Auxotrophy testing

For auxotrophy testing, 5 µl of a 107 spores/ml suspension were applied to the middle of a minimal medium plate with or without supplements. As minimal medium, MAM without peptone and glucose as carbon source was used. Plates were incubated at 30 °C for 1 week.

Plasmid constructions

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 Fisher Scientific Inc., Waltham, MA, USA) and verified by sequencing (at Microsynth, Balgach, Switzerland). The fragments were released for subsequent cloning purposes by digestion with suitable restriction endonucleases (NEB).

Table 2 Primers used in this study

For the construction of pCD-Δhis1, the 5′ flank was amplified by PCR using the primers 80820_5fwd-BspEI and 80820_5rev-EcoRI and chromosomal DNA of T. reesei QM6a Δpyr4 and inserted into pJET-pyrG [13]. Next, the partial direct repeat of the 5′flank was fused to the 3′flank by a splicing by overlap extension PCR. The fragments were amplified using the primers 80820_5fwd2-AflII and 80820_5rev-SOE or 80820_3fwd-SOE and 80820_3rev-NsiI. The fusion PCR fragment was inserted into the latter plasmid yielding pCD-Δhis1 (Fig. 1A; Additional File 6).

For the construction of pCD-ReHis1, the 5′flank of his1 was amplified with the primers 80820_5fwd-BspEI and 80820_5rev-MCS and inserted into pJET1.2 (Thermo Fisher Scientific) in the opposite direction of the eco47IR gene. Next the promoter and the coding region of his1 was amplified with the primers P80820_fwd-MCS and 80820_3rev-ClaI and inserted into the latter plasmid via NheI and ClaI. The resulting plasmid pCD-ReHis1 (Additional File 7) contains a multiple cloning site (BamHI, EcoRI, NheI, NdeI, PstI) between the 5’flank and the promoter of his1 to facilitate insertion of further genes.

For the construction of pCD-ReHis1-eyfp (Additional File 8), the expression cassette for EYFP, containing the constitutive pki promoter, a codon-optimized eyfp gene, and the cbh2 terminator, was amplified with the primers Ppki_fwd-BamHI and Tcbh2_rev_NheI using pCD-EYFP [10] as template and inserted into pCD-ReHis1 via BamHI and NheI.

For the construction of the standard plasmids for the qPCR assay to determine the copy number of eyfp, a part of the cbh1 coding region and the eyfp expression cassette were amplified using the primers cbh1_fwd_qPCR and cbh1_rev_qPCR, and Ppki_fwd-Kpn2I and Tcbh2_rev_PstI, and chromosomal DNA of QM6a Δpyr4 and pCD-EYFP [10] as template, respectively, and inserted into pJET1.2 (Thermo Fisher Scientific).

Fungal transformations

The protoplast generation and polyethylene glycol mediated transformation of T. reesei was performed as described previously [14]. Typically, 15 µg of linearized plasmid DNA (digested with NotI, precipitated with ethanol, resuspended in 15 µl sterile ddH2O) was used for the transformation of 107 protoplasts (in 100 µl). Selection was described previously [10]. Resulting candidates were subjected to homokaryon purification by streaking conidia on plates with selection medium containing 0.1% (w/v) Igepal CA-630 (Sigma-Aldrich, part of Merck KGaA, Darmstadt, Germany).

Marker recycling

For the minimal effort marker recycling, the strain T. reesei QM6a Δhis1(pyrG  +) was incubated on MAM plates without peptone containing uridine, histidine, and 5-FOA. The plate was incubated at 30 °C for up to 4 weeks, until the pyrG marker was lost due to a random internal homologous recombination (Fig. 1B) and the fungus gained 5-FOA tolerance (Additional File 2).

Isolation of chromosomal DNA

Chromosomal DNA was isolated from mycelium by grinding in liquid nitrogen followed by a phenol/chloroform extraction [14]. RNA was degraded using RNaseA (Thermo Fisher Scientific). DNA was precipitated with isopropanol, washed with 70% ethanol, and dissolved in ddH2O.

Genotype testing by PCR

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 1 kb Plus DNA Ladder (NEB) was used as standard.

Determination of the eyfp copy number

Dilutions of the chromosomal DNA of the EYFP-expressing strains were used as template in a qPCR assay targeting the eyfp and the cbh1 gene. For comparison, plasmids carrying the target size were used. The relative copy number of eyfp in relation to cbh1 was calculated (Additional File 3) using the Pfaffl method [15].

RNA extraction

Fungal strains were cultivated in Erlenmeyer flasks for 48 h, mycelia and supernatants were separated by filtration through Miracloth (Merck Millipore, part of Merck KGaA, Darmstadt, Germany). Approx. 0.05 g of harvested mycelia were resuspended in 1 ml RNAzol RT (Sigma-Aldrich) and lyzed using a Fast-Prep-24 (MP Biomedicals, Santa Ana, CA, USA) with 0.37 g of small glass beads (0.1 mm diameter), 0.24 g of medium glass beads (1 mm diameter), and a single large glass bead (5 mm diameter) at 6 m/s for 30 s. Samples were incubated at room temperature for 5 min and then centrifuged at 12,000g for 5 min. 750 µl of the supernatant were mixed with 750 µl ethanol and RNA isolated using the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. This Kit includes a DNAse treatment step. The concentration and purity were measured using the NanoDrop ONE (Thermo Scientific).

Transcript analysis by RT-qPCR

500 ng of isolated total RNA was reverse transcribed using the LunaScript RT SuperMix (NEB) according to the manufacturer’s instructions. The resulting cDNA was diluted 1:50 and 2 µl were used as template in a 20 µl reaction using the Luna Universal qPCR Master Mix (NEB) according to the manufacturer’s instructions. All reactions were performed in technical duplicates on a Rotor-Gene Q system (Qiagen, Hilden, Germany). Calculations of the relative transcript levels were performed according to the Pfaffl method [15] using the reference genes sar1 and act1 for normalization according to [16].

Fluorescence measurements

The strains were cultivated in fluorescence 96 well plates in technical triplicates in two independent experiments, and in parallel in technical triplicates in a transparent 96 well plates to determine the optical density. After 72 h cultivation the total fluorescence or the optical density of the cultures was measured in a Glomax Multi Detection System (Promega, Madison, WI, USA) using the blue filter kit (excitation peak wavelength at 490 nm, emission wavelengths between 510 and 570 nm) or absorbance at 600 nm.

Availability of data and materials

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

Abbreviations

5-FOA:

5-Fluororotic acid

AICAR:

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-Monophosphate

EYFP:

Enhanced yellow fluorescence protein

MAM:

Mandels-Andreotti medium

PCR:

Polymerase chain reaction

RT-qPCR:

Quantitative reverse transcription PCR

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Acknowledgements

Not applicable.

Funding

This study was supported by the Austrian Science Fund (FWF, https://www.fwf.ac.at/) (P 34036 to CD).

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Authors

Contributions

PP performed the genotype and phenotype testing, cultivation experiments, the enzyme, and RT-qPCR assays and co-drafted this manuscript, MJ was involved in plasmid and strain design and construction, RLM and AMA provided resources for this study and revised the manuscript, CD designed this study, was involved in plasmid and strain construction, supervised the experiments, and co-drafted this manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Christian Derntl.

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Supplementary Information

Additional file 1:

Genotype verification of the constructed strains.

Additional file 2:

Marker recycling due to a spontaneous internal recombination leading to the loss of the pyrG gene.

Additional file 3:

Determination of the copy number of the integrated eyfp gene.

Additional file 4:

Growth curves of the T. reesei strains QM6a Δpyr4, Δpyr4 eyfp (his1), eyfp (pyr4), and eyfp (asl1) on different carbon sources in a 96-well plate.

Additional file 5:

Raw data and calculation for the fluorescence units of the T. reesei strains QM6a Δpyr4, Δpyr4 eyfp (his1), eyfp (pyr4), and eyfp (asl1) on different carbon sources in a 96-well plate.

Additional file 6:

Genomic sequence of pCD-Δhis1.

Additional file 7:

Genomic sequence of pCD-ReHis1.

Additional file 8:

Genomic sequence of pCD-ReHis-eyfp.

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Primerano, P., Juric, M., Mach, R. et al. Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei. Fungal Biol Biotechnol 8, 9 (2021). https://doi.org/10.1186/s40694-021-00116-5

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Keywords

  • Trichoderma reesei
  • Histidine auxotrophy
  • ATP phosphoribosyltransferase
  • Marker recycling
  • Gene targeting
  • Heterologous expression