A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)
© Jørgensen et al.; licensee BioMed Central Ltd. 2014
Received: 4 September 2013
Accepted: 10 February 2014
Published: 6 March 2014
The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering.
In this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the T. reesei genome ensuring that enzyme production from different transformants can be directly compared. The ade2 locus was selected as integration site since ade2 mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a tku70 disruption to increase gene targeting efficiency and a new bidirectional marker, pyr2, for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%.
The new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of T. reesei and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with ade2 as integration site was confirmed by expression of a Thermomyces lanuginosus lipase.
Trichoderma reesei is a key workhorse for commercial scale production of different enzymes used by the bioethanol, pulp, paper, and textile processing industries. This status is a consequence of its unique capability of producing and secreting large amount of enzymes, its amenability to large-scale fermentation as well as its long history of safe use in industrial enzyme production. Importantly, production of several T. reesei enzymes has obtained the generally recognized as safe (GRAS) status by the U.S. Food and Drug Administration (FDA). Moreover, T. reesei serves as a model organism for the regulation of expression and biochemistry of (hemi-)cellulose degradation enzymes and pathways [1, 2]. There is an increasing demand for these enzymes since they are employed for the saccharification of cellulosic plant biomass to simple sugars for biofuel production. As enzymes constitute an important cost in the production of bioethanol, large research efforts as well as large government funding have aimed to continuously improve T. reesei as an enzyme production host. This process will be greatly facilitated by the fact that its genome has recently been sequenced setting the stage for strain development by directed genetic engineering .
Unfortunately, the highly efficient protein synthesis machineries of T. reesei, which enable yields of homologous proteins in excess of 100 g/l , has so far not prevailed for synthesis of heterologous proteins and yields remain low . Methods and tools to improve synthesis of heterologous proteins in this fungus are therefore highly desirable.
One bottle neck towards this goal has been the low efficiency of gene targeting in T. reesei, but like in a number of other fungi this problem has been dramatically reduced by deleting a gene involved in Non-Homologous End-Joining (NHEJ). In such strains the frequency of successful integration by gene targeting was first reported to increase to >95% from the 5-10% obtained with wild-type strains . However, there is still room for improvement as a recent study in T. reesei has demonstrated that the efficiency of homologous integration in NHEJ deficient strains can be highly site specific and may vary from 33 to 100% depending on insertion site .
Before T. reesei can be routinely used for heterologous protein production, it is necessary to gain insights into e.g. the influence of promoter sequences, effects of codon optimized synthetic genes, or expression rates of selected orthologous genes. This type of analysis requires construction of large numbers of strains where the genes to be compared are expressed from a defined locus in isogenic strains to allow for proper comparisons. Moreover, such analysis may often include experiments that require multiple rounds of genetic engineering in the same strain. Consequently, the need for molecular tools that allow easy genetic modifications of T. reesei for industrial strain development are urgent. We have therefore developed a new expression platform in T. reesei that facilitates heterologous gene expression from a defined locus with an improved gene-targeting throughput. Our expression platform is composed of four parts: 1) a versatile integration plasmid for gene expression containing a USER (Uracil-Specific Excision Reagent) cassette for highly efficient ligase-free USER cloning, 2) a bidirectional marker pyr2, encoding orotate phosphoribosyl transferase, that allows for iterative gene targeting, 3) a tku70 gene disruption strain for efficient gene targeting, and 4) a color marker that facilitates identification of correctly targeted strains, even in early stages of colony development. The use of pyr2 as a bidirectional marker has not previously been reported for T. reesei, but we here demonstrate that it serves as a highly reliable alternative to the previously used pyr4 gene , encoding orotidine-5′-monophosphate decarboxylase. The applicability of the expression system was confirmed by expression of a Thermomyces lanuginosus lipase as reporter protein.
Results and discussion
Construction of a tku70∆ pyr2∆ strain
First, the tku70 gene involved in NHEJ was disrupted in the original T. reesei QM6a isolate by transforming this strain with a gene-targeting substrate based on the amd S marker; see Materials and methods for details. Sixty transformants were obtained and a subsequent PCR analysis (data not shown) identified a single transformant containing the desired disruption of tku70. The correct integration of amdS into tku70 in this transformant was confirmed by Southern blotting (Additional file 1: Figure S1) and the resulting strain was named MJ-T-001.
pyr2 as a bidirectional marker
To demonstrate that pyr2 can be used as a marker for iterative gene targeting, we transformed MJ-T-013 with a gene targeting substrate designed to delete ade2. Nine uracil prototrophic transformants appeared on the primary transformation plates after transformation. Of these nine transformants, four developed orange/red coloration after four days of growth, indicating successful deletion of the ade2 gene (Figure 3). All nine colonies were tested for adenine auxotrophy and as expected, the four red colonies were all adenine auxotrophs whereas the remaining five were not. Correct deletion of ade2 in the four selected red transformants was confirmed by PCR (data not shown). One of the four transformants containing the pks4 and ade2 double deletion was verified by Southern blotting (Figure 1) and named MJ-T-021. Successful construction of pks4 and ade2 double deletion strains demonstrate that pyr2 can be used as an alternative to pyr4 for iterative gene targeting. Importantly, the coding sequence of pyr2 is 435 bp shorter than that of pyr4, and as a consequence episomal vectors and gene targeting substrates that are based on pyr2 will be smaller than if they are based on pyr4, which is often advantageous.
A versatile integration vector for gene expression at ade2
Growth rates of mutant strains in MM supplemented with 0.5 mM adenine
pyr2 Δ ade2 Δ::pyr2
pyr2 Δ pks4 Δ ade2 Δ::pyr2
Growth rate (mm/h)
0.91 (st.dev. 0.04)
0.99 (st.dev. 0.02)
0.91 (st.dev. 0.09)
To demonstrate this concept we decided to express the T. lanuginosus lipase gene, lip, from the ade2 locus. Accordingly, lip was positioned between the A. nidulans PgpdA promoter and TtrpC terminator in pMJ-023, resulting in pMJ-051. The gene-targeting substrate containing the PgpdA::lip::TtrpC fragment was liberated from the vector backbone by restriction enzyme digestion and inserted into ade2 using pyr2 as selection marker. After transformation, several red colonies appeared and correct insertion of the expression construct in three of these transformants was confirmed by PCR and Southern blot analyses (Additional file 3: Figure S3). The three transformants, MJ-T-033-1, -2 and −3, were analyzed for T. lanuginosus lipase production using a well-developed assay for measuring lipase activity, see Materials and methods. In a parallel experiment, an empty expression construct containing PgpdA::TtrpC was inserted into ade2 to generate strain MJ-T-020, which was analyzed in the same manner. The average lipase activities (Lipase Units, LU) for MJ-T-020, MJ-T-033-1, -2 and -3 were 1.19 LU ± 0.05, 2.00 LU ± 0.15, 1.85 LU ± 0.09 and 2.09 LU ± 0.15 pr. 10 μl supernatant, respectively. Hence, a significant and reproducible increase in the amounts of lipase activity could be detected in the strains expressing the PgpdA controlled lip. Specifically, additional lipase activity corresponding to an average of 0.79 LU was produced in these strains.
In summary, we have developed an expression platform, which is designed for high-throughput construction of defined integrated T. reesei strains suitable for setup of large expression studies. The compatibility of USER cloning with PCR enables easy site-directed mutagenesis, promoter swaps, and epitope- and GFP tagging for protein engineering, characterization, purification, and production. Moreover, selected mutants can be further analyzed/optimized by iterative gene targeting using pyr2 as a bidirectional selective marker.
Materials and methods
Strains and media
Overview of the strains used in this study
Growth in presence of 5-FOA
(Martinez et al., 2008) 
tku70 -::amd S+
tku70 -::amd S+ pyr2 Δ
tku70 -::amd S+ pyr2 Δ ade2 Δ::pyr2
tku70 -::amd S+ pyr2 Δ pks4 Δ::pyr2
tku70 -::amd S+ pyr2 Δ pks4 Δ
tku70 -::amd S+ pyr2 Δ ade2 Δ::PgpdA-TtrpC-pyr2
tku70 -::amd S+ pyr2 Δ pks4 Δ::pyr2 Δ ade2 Δ::pyr2
tku70 -::amd S+ pyr2 Δ ade2 Δ::PgpdA-lip- TtrpC-pyr2
The putative T. reesei proteins encoded by ade2 (JGI ID: 105832), pyr2 (JGI ID: 21435) and pks4 (JGI ID: 82208) were identified by BLASTing i) the 250 amino acid residues (aa) of the A. oryzae PyrF protein (/EMBL/GenBank protein accession number XM_001821908.2), ii) the 2157 aa sequence of the A. nidulans Ywa1 protein and iii) the 571 aa sequence of the S. cerevisiae Ade2 protein, respectively, in the JGI T. reesei filtered model proteins database (http://genome.jgi-psf.org/pages/blast.jsf?db=Trire2). The corresponding gene sequences were subsequently extracted from the same source.
An overview of the primers used for vector construction and the resulting plasmids are listed in Additional file 5: Table S2 and Additional file 6: Table S3, respectively. PCR fragments used for the constructions were made by using PfuX7 polymerase  and purified from agarose gel using the GFX kit (GE healthcare, Little Chalfont, United Kingdom). Specifically, pMJ-001 was made by inserting the relevant PCR fragment (see Additional file 7: Figure S4 and Additional file 5: Table S2) into pCR2.1 (Invitrogen) by topoisomerase-mediated ligation according to the instructions provided by the manufacturer. pMJ-005 was made by inserting the relevant PCR fragment into a StuI and SnaBI vector fragment of pMJ-001 using In-Fusion® ligation (Clontech). The remaining vectors were made by fusing PCR fragments with the pU1111-1 vector backbone  by restriction enzyme and ligase independent uracil-excision cloning  using the USER™ Friendly Cloning Kit protocol (New England Biolabs), see Additional file 5: Table S2 and Additional file 6: Table S3 for details. All vectors were cloned by transformation into chemically competent Fusion-Blue E. coli cells (Clontech, Mountain View, USA) as described in the In-Fusion® Dry-Down PCR Cloning Kit Protocol-at-a-Glance.
T. reesei protoplastations and transformations were performed as described by Gruber and co-workers . Approximately 10 μg linearized DNA by restriction endonucleases was used for each transformation. Correct targeted integration was verified by diagnostic PCRs and Southern blots; see primers in Additional file 8: Table S4. All strains were incubated on solid media for five days at 28°C; and in liquid media for 48 hours at 30°C and 200 RPM. The exception was for lipase assays as described later.
The gene targeting substrate for construction of MJ-T-001 was obtained by digesting pMJ-005 with ClaI. This fragment was transformed into QM6a and transformants selected by using the amdS selection marker on MM plates supplemented with acetamide. All remaining gene-targeting substrates were liberated from the plasmid backbone by NotI digestion and gel purified. MJ-T-006 was generated by transforming MJ-T-001 with the gene targeting substrate obtained from pMJ-017. In this case transformants were selected by plating on MM plates containing 5-FOA and uridine. MJ-T-009 and MJ-T-012 was obtained by transforming MJ-T-006 with gene targeting substrates isolated from pMJ-031 and pMJ-030, respectively. MJ-T-009 was selected on MM supplemented with adenine and MJ-T-012 on MM. MJ-T-013 was generated from MJ-T-012 by selecting for recombinants where the pyr 2 marker has been lost by direct repeat recombination. Specifically recombinants were selected by plating 108 spores on 5-FOA MM plates.
The pyr2 loop out strain, MJ-T-013, was transformed with the gene targeting substrate isolated from pMJ-031 and transformants selected on MM plates to acquire MJ-T-021. MJ-T-020 was obtained by transforming MJ-T-006 with the gene targeting substrate isolated from pMJ-023 and plating on MM plates. After transforming MJ-T-006 with the gene targeting substrate originating from pMJ-051, MJ-T-033-1, -2 and −3 were isolated from MM plates.
All transformants were streak-purified on the proper selection medium before further analysis. All strains were verified by diagnostic PCR and Southern blot, Figure 1, Additional file 1: Figure S1, Additional file 3: Figure S3 and Additional file 8: Table S4.
Growth rate measurements
The linear growth rates of MJ-T-001, MJ-T-009, MJ-T-012 and MJ-T-021 were measured in race tubes, as described by White and Woodward .
Expression of T. lanuginosus lipase from the ade2 site
The T. lanuginosus lipase gene (lip) (accession no. AF054513) was kindly provided by Jan Lehmbeck, Novozymes, Bagsvaerd. The lipase was produced by growing MJ-T-033-1, -2 and -3 in 10 ml YPD at 30°C for four days at 200 rpm. At this point, the amount of lipase in the medium was determined by measuring the esterase activity. Specifically, the rate of p-nitrophenol formation using p-nitrophenyl valerat as substrate was measured in microtiter plate wells by mixing 10 μl supernatant, 20 μl dilution buffer (50 mM Tris/HCL (pH 7.5), 10 mM CaCl2 and 0.075% Brij-35 (Thermo Fisher Scientific, Rockford, USA)) and 200 μl substrate solution (0.6 mM 4-Nitrophenyl valerate (Sigma N4377) dissolved in methanol).
The Lipase Units (LU) were measured in an ELISA reader as absorption at 405 nm (peak absorbance of p-nitrophenol) in 30-second intervals for 40 minutes. The amount of LU in the samples was calculated based on included standards.
This study was supported by grant 09–064967 from the Danish Council for Independent Research, Technology, and Production Sciences to UHM. MSJ’s Ph.D project was funded by Novozymes A/S, the Department of Systems Biology (DTU) and the Danish research council via the Research program for Biotechnology (FOBI), Denmark.
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