I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei
Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
KeywordsMeganuclease DNA repair Counter selection Targeted integration Hypocrea jecorina
Trichoderma reesei (teleomorph Hypocrea jecorina) can secrete large amounts of extracellular protein (up to 100 g/L), which makes it a paradigm host for homologous and heterologous protein production (Anderson et al. 2013; Schuster and Schmoll 2010). To improve production, activity or other properties of industrially interesting enzymes, both random and systematic approaches to generate enzyme variants are employed (Adrio and Demain 2014; Turner 2009). To simplify comparison among enzyme variants or different homologues expressed in filamentous fungi and, in particular T. reesei, it is highly desirable that in order to ensure an identical genetic environment, the corresponding DNA constructs are integrated at a defined locus in the genome. Random integration of expression cassettes often leads to significant variation in production levels caused by differences in copy number and/or sites of integration. Therefore, it has been attempted to increase the efficiency of gene targeting in T. reesei by several means, such as by increasing the size of the region homologous to the target locus (Catalano et al. 2011) or by developing strains that are deficient in non-homologous end joining (NHEJ) (Catalano et al. 2011; Guangtao et al. 2009). However, even though the efficiency of homologous integration events is tremendously increased in strains deficient in NHEJ, it remains highly locus-dependent (Schuster et al. 2012). Transformation frequencies with NHEJ-deficient strains are often low, making it difficult and laborious to generate enough mutants for library screening. Consequently, there is still a need to develop alternative methods for efficient gene targeting into the genome of T. reesei that lead to high homogeneity in protein expression among transformants. Another limiting factor associated with T. reesei transformation is a relative low frequency of stable, DNA integration events. A substantial number of primary transformants (30–50 %) are abortive ones, in which a plasmid is only transiently expressed and not integrated into the genome.
In this study, we present an approach that not only increases the number of stable transformants in T. reesei up to sixfold but also increases the efficiency of targeted integration of the gene of interest. The approach is based on stimulating the formation of a DNA double-strand break (DSB) at a specific locus of the genome, which is subsequently efficiently repaired by the receiving organism to maintain its genomic integrity and survival (Bollag et al. 1989; Szostak et al. 1983). Eukaryotic cells repair DSBs either by the NHEJ pathway or via homology directed repair (HDR) with homologous sequences flanking the DSBs or any other available homologous template, including exogenous donor sequences (Cahill et al. 2006). Genomic DSBs can occur either naturally or be induced artificially. It has been shown that DSBs can be specifically induced in eukaryotic genomes by using the yeast I-SceI endonuclease (Arazoe et al. 2014; Choulika et al. 1995; Kuijpers et al. 2013; Puchta et al. 1993; Rouet et al. 1994). I-SceI is a mitochondrial homing endonuclease encoded by the Saccharomyces cerevisiase mitochondrial genome and recognizes a 18-basepair-long DNA sequence (5′-TAGGGATAACAGGGTAAT-3′) (Monteilhet et al. 1990; Plessis et al. 1992; Fairhead and Dujon 1993). Since the recognition site is rather long and specific, I-SceI recognition sites are absent in most eukaryotic genomes, including the T. reesei genome. Expression of the endonuclease I-SceI has been previously demonstrated to efficiently induce DSBs at I-SceI sites inserted at specific sites in prokaryotic cells (Meddows et al. 2005) and also in several eukaryotes, including mammalian cells (Choulika et al. 1995), plants (Puchta et al. 1993) and unicellular eukaryotes (Glover and Horn 2009). In filamentous fungi, I-SceI-mediated DSBs have been recently described in Pyricularia oryzae (Arazoe et al. 2014), a fungus belonging to the class of Sordariomycetes, to which T. reesei also belongs. In the present report, we have introduced I-SceI recognition sites into the genome of T. reesei and have investigated the effect of DSBs mediated by heterologous expression of I-SceI on transformation efficiency, on frequency of targeted integration of a reporter gene and on the expression levels of the reporter protein. We have also shown that about two thirds of the transformants integrated a reporter T. reesei glucoamylase gene at a pre-assigned locus and expressed it uniformly.
Materials and methods
Strains and cultivation conditions
Fungal strains and plasmids used in this study
Genotype or description
Source or reference
Anderson et al. 2013
pyr4− and PcbhI−
DuPont bioscience. Leiden, The Netherlands
P37∆cbhIpyrG-26 with I-SceI restriction site cassette integrated at cbh2 locus
Carries a I-SceI restriction site cassette
Contains the full gene of A. nidulans pyrG
cbhI promoter and amdS selection
I-SceI under control of the inducible cbh1 promoter with amdS selection
Carries the T. reesei glucoamylase gene
Carries a T. reesei glucoamylase cassette with homologous regions of the I-SceI landing sites cassette (pBJP6)
T. reesei was transformed using the polyethylene glycol (PEG)-mediated protoplast transformation protocol described by Penttilä et al. (1987) with slight modifications. Fifty-millilitre cultures inoculated with 5 × 108 conidia were grown in the dark at 30 °C and 200 rpm for 12–20 h. A total of 675 mg lysing enzyme (Sigma-Aldrich, Zwijndrecht, The Netherlands) were dissolved in 15 mL of 1.2 M MgSO4-10 mM sodium phosphate buffer, pH 5.8. Protoplasting was performed at 25 °C and 90 rpm and was verified every 30 min by microscopy. Two hundred microlitres of protoplast suspension was mixed with 5–10 μg of DNA and 2 mL of freshly made PEG buffer. Stable transformants were obtained by streaking on TrMM plates containing the required selection pressure, for two successive rounds. Single colonies obtained after double streaking were selected for sporulation and further analysis.
T. reesei liquid cultures were grown in 24-well plates configured such as to release lactose from a solid, porous matrix. Each well contained 1.25 mL of an NREL medium (9 g/L casamino acids, 5 g/L (NH4)2SO4, 4.5 g/L KH2PO4, 1 g/L MgSO4·7H2O, 1 g/L CaCl2·2H2O, 33 g/L PIPPS buffer, at pH 5.5, 0.25 % T. reesei trace elements, as described above). Escherichia coli DH5α strain was used for plasmid construction and propagation using standard techniques.
DNA manipulations and molecular analyses
Genomic DNA extraction of T. reesei, diagnostic PCR and Southern blot analysis were performed as previously described (Meyer et al. 2010). Restriction enzymes and DNA dephosphorylation and ligation kits were obtained from Invitrogen (Bleiswijk, The Netherlands) or Thermo Fisher (Leusden, The Netherlands) and used according to the instructions of the manufacturer. Sequencing was performed by Macrogen (Amsterdam, The Netherlands).
Construction of the I-SceI restriction site cassette
List of primers used in this study
Sequence (5′ to 3′oriented)
Construction of the I-SceI expression vector
A codon-optimized gene coding for the Saccharomyces cerevisiae I-SceI sequence (SGD ID S000007279) was synthesized for expression in T. reesei (Geneart, Regensburg, Germany). The nucleotide sequence encoding I-SceI has been deposited in GenBank under accession number KR584660. To construct a I-SceI expression vector, the I-SceI gene was cloned via a Gateway recombination into the telomeric plasmid pTTT (Aehle et al. 2011), where I-SceI expression was driven by the T. reesei cbhI-inducible promoter. A I-SceI variant in which we included the nuclear localization signal (NLS) (MATPSSVASS SSRDQVQRIH RVTRENRHLW YQLTVLQQPE RARACGSG) of the T. reesei Velvet protein (JGI ID 122284) was constructed and tested in parallel. To identify this putative NLS sequence in the T. reesei Velvet protein, the putative NLS sequence identified in the Velvet protein of A. nidulans (Stinnett et al. 2007) was used for alignments.
Construction of the glucoamylase expression cassette for targeted integration
The plasmid pTrex6g-GA (Bower et al. 2012), which harboured the wild-type glucoamylase gene of T. reesei under control of the cbhI promoter, was used to construct the glucoamylase expression cassette for integration at the I-SceI landing sites. To allow homologous integration of this reporter, the cbh2 terminator region (Tcbh2) was cloned in pTrex6g-GA. The Tcbh2 was amplified by PCR using the primers GSP5 and GSP6 (Table 2) and the genomic DNA of the T. reesei QM6a WT strain. The PCR product was digested with AsiSI and cloned into the same restriction sites of pTrex6g-GA, to form plasmid pJP8. The 10-kb glucoamylase expression fragment was cut out from pJP8 with PsiI and used for transformation.
In vivo analysis of I-SceI activity in T. reesei and fluorescent microscopy
Purified T. reesei transformants that carried I-SceI restriction sites and the I-SceI expression cassette were point inoculated on TrMM with 2 % of glucose or lactose as a carbon source with or without addition of uridine to the medium. The activity of I-SceI was monitored by formation of sectors without growth after incubating transformants for several days at 30 °C. Sector formation is likely to be the consequence of excision of the pyrG marker and subsequent repair of the genomic DNA induced by the I-SceI-mediated DSB. Transformants that formed sectors on solid medium were selected for GFP expression under fluorescence microscopy. A total of 107 conidia of sector-forming transformants were inoculated in 5-mL germination medium containing 2 % glucose/sophorose (30:1) for induction and supplemented with 0.003 % yeast extract and 10 mM uridine, and cultivated on cover slips for 28 h at 30 °C. Samples were observed with Axioplan 2 fluorescence microscope (Zeiss, Sliedrecht, The Netherlands) equipped with a DKC-5000 digital camera (Sony) using different contrast or GFP settings. Images were captured and processed using Adobe Photoshop 6.0 (Adobe Systems Inc.).
Quantification of the efficiency of pyrG excision mediated by I-SceI expression
To determine the frequency of the pyrG marker loss as a consequence of I-SceI expression, the strain JP7.7.12 bearing the I-SceI restriction sites and the I-SceI expressing cassette (pTTT-ISceI) was used. As a control, strain JP7.7_pTTT bearing the I-SceI restriction sites and the pTTT plasmid without I-SceI was included. About 100 spores of each strain harvested from minimal medium without uridine were grown separately under inducing conditions (TrMM with 2 % lactose as carbon source) supplemented with uridine. Addition of uridine into the medium allowed for growth of all spores including those that will lose the pyrG marker under I-SceI expression conditions. To quantify the frequency of the pyrG loss events, single colonies from I-SceI-induced medium were transferred into minimal medium with and without uridine. The efficiency of the marker excision was determined as the ratio of the number of pyrG-negative colonies and the total number of colonies grown on the plate with uridine.
Determination of glucoamylase activity and glucoamylase levels
Purified transformants were grown in production medium for 5 days and glucoamylase was measured from the culture filtrate. The activity of glucoamylase was determined using Betamyl as a substrate (Megazyme International, Bray, Ireland). Ten microlitres of the culture samples containing glucoamylase were mixed with 90 μL of Betamyl diluted in 50 mM sodium acetate pH 4.8. The reaction is performed at 37 °C for 20 min and quenched with 50 μL of 1 M sodium carbonate pH 9. Activity was monitored by the release of p-nitrophenol measured colorimetrically at 405 nm. For each transformant, the glucoamylase activity was measured in triplicate. Glucoamylase levels were detected using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed under denaturing conditions according to Laemmli (1970). Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Veenendaal, The Netherlands).
Design of a marker excision method to monitor I-SceI activity
Construction of a T. reesei strain harbouring the I-SceI restriction sites at the cbh2 locus
The 7.5-kb reporter cassette as described above was linearized and introduced into a T. reesei strain deleted for cbhI. Transformants were analysed by Southern blot for correct integration at the cbh2 locus. Southern blot analysis revealed three transformants harbouring a single copy of the complete I-SceI cassette at the cbh2 locus (JP7.7, JP7.9 and JP7.12) (Fig. S2 in the Supplementary Material). Other transformants either had multiple copies of the cassette integrated (JP7.10, JP7.11, JP7.13, JP7.14 and JP7.15) or did not seem to insert the complete cassette into the genome (JP7.8) (Fig. S2 in the Supplementary Material). Transformant JP7.7 was used in subsequent experiments.
I-SceI expression in T. reesei induces DSB at the targeted chromosomal locus
The frequency of the I-SceI-mediated excision was determined by comparing the frequency of losing the pyrG marker in the I-SceI-expressing and I-SceI-non-expressing strains (strains JP7.7.12 and JP7.7_pTTT, respectively). Therefore, about 100 spores of each strain were plated on lactose-containing induction medium supplemented with uridine. After growth for 3 days, each colony formed was further scored for its growth in the absence of uridine. Sixty-six percent of the colonies of the JP7.7.12 strain bearing the I-SceI restriction sites and containing the I-SceI expression cassette (pTTT-ISceI) were found to be auxotrophic for uridine, whereas only 1.3 % of the colonies of JP7.7_pTTT strain bearing the empty pTTT plasmid were uridine-dependent. Thus, a high frequency of pyrG excision mediated by in vivo-expressed I-SceI nuclease was observed during a plate growth test.
I-SceI-mediated DSB increases transformation efficiency and transformant stability
The effect of I-SceI induction on number and stability of transformants
# of primary transformantsa
% stable transformantsb
% Gla-positive transformantsc
% Gla-positive pyrG − transformantsd
Homologous recombination efficiencye
17/32 (53 %)
21/36 (58 %)
12/40 (30 %)
2/12 (16 %)
2/12 (16 %)
45/50 (90 %)
22/40 (55 %)
15/22 (68 %)
15/22 (68 %)
22/50 (44 %)
15/40 (37 %)
7/15 (46 %)
7/15 (46 %)
We further analysed the stability of the transformants obtained in each group of transformation, as by subsequent purification on MM-chlorimuron ethyl plates. Ninety percent of the transformants obtained after the induction of I-SceI were stable against 44 and 58 % of colonies grown under repressed conditions and for the control strain, respectively (Table 3). This indicates that heterologous expression of the I-SceI nuclease increased transformation efficacy and transformant stability. Instability of transformants usually results from a lack of integration of a given DNA cassette into the genome and has been previously described to be a major bottleneck for T. reesei transformation (Jørgensen et al. 2014).
Uniform expression of the reporter glucoamylase gene after targeted integration at the engineered genomic locus
Rare-cutting, double-stranded DNA endonucleases, called meganucleases, have emerged as a powerful tool for genome manipulation. Several studies have demonstrated that expression of such endonucleases in prokaryotic and eukaryotic cells stimulated homologous recombination between a given repair template and a genome locus containing an endonuclease recognition site (Choulika et al. 1995; Glover and Horn 2009; Meddows et al. 2005; Rouet et al. 1994). The aim of the current study was to increase efficiency of the gene-targeted integration at a predetermined locus in the T. reesei genome to ensure high expression levels and limited variability of protein expression. To this end, the S. cerevisiae endonuclease I-SceI was expressed in T. reesei and I-SceI recognition sites flanking a reporter construct were introduced into the cbh2 locus of T. reesei. We demonstrated that expression of I-SceI resulted in endonuclease activity. The I-SceI protein appeared to be imported into the T. reesei nucleus via the intrinsic NLS signal, as we did not detect obvious differences in efficiency of marker excision between the original I-SceI protein and a I-SceI variant fused to a nuclear targeting signal of the T. reesei Vel1 protein. We analysed the impact of the I-SceI-mediated DSBs on transformation frequency, transformant stability and efficiency of homologous recombination.
Our data indicate that DSB mediated by I-SceI in T. reesei improves the efficiency of transformation and the stability of the transformants and promotes gene targeting at a defined locus. At least sixfold more transformants were obtained when I-SceI was induced, and, more importantly, 90 % of them were found to be stable. Genetic instability is a major bottleneck in T. reesei transformation and usually is a consequence of a lack of integration of a transformed DNA cassette into the genome or due to a tandem integration of multiple copies, which could be excised through a loop-out event (Aw and Polizzi 2013; Jørgensen et al. 2014; Le Dall et al. 1994; Lee and Da Silva 1997; Ohi et al. 1998). This is the first study that addresses issues related to the stability of transformants mediated by I-SceI in filamentous fungi and particularly in the industrial fungus T. reesei. Furthermore, when I-SceI was expressed in the cells, the frequency of homologous recombination increased up to 68 %.
A recent study of gene targeting based on I-SceI-induced DSBs in P. oryzae (Magnaporthe oryzae) reported that in this fungus, I-SceI expression increases targeted integration up to about 40 %, which is comparable to our findings (Arazoe et al. 2014). Targeted integration can be also improved in filamentous fungi by deletion of the genes involved in non-homologous end joined (NHEJ) recombination (Ninomiya et al. 2004; Krappmann et al. 2006; Meyer et al. 2007; Guangtao et al. 2009; Steiger et al. 2011). However, inactivation of the NHEJ genes usually increases strain sensitivity towards chemicals and physical DNA-damaging agents and mostly reduces efficiency of transformation as compared to a strain with intact NHEJ genes (Zhang et al. 2011). In addition, targeted integration in strains deficient of NHEJ proteins can be highly locus-dependent and may vary from 33 to 100 % depending on the insertion site (Jørgensen et al. 2014; Schuster et al. 2012). Our study shows that using the I-SceI endonuclease, we can improve targeted integration events in a T. reesei strain with intact NHEJ genes. Moreover, the targeted integration transformants could be specifically selected by adding 5-fluoroorotic acid (5′FOA) in the transformation medium. About 90 % of the pyrG− transformants expressed glucoamylase, indicating that the DNA cassette was integrated at the intended locus. Moreover, Southern blot analysis confirmed the presence of a single copy of the expression fragment in these transformants. Importantly, we found that glucoamylase production levels were much more consistent among the transformants that harboured the expression cassette at the targeted locus as compared to transformants with a random integration pattern. Homogeneity in protein expression in the population of transformants is always a great challenge in the field of fungal biotechnology because of the low efficiency of targeted integration events (Aw and Polizzi 2013). Thus, our technique may be employed for high-throughput screening of enzyme libraries or for construction of production strains with predetermined integration sites, which is especially relevant in industrial biotechnology.
We thank Prof. Dr. Paul Hooykaas and Dr. Jaap Visser for the helpful discussions. We thank Dr. F. Klis for carefully reading the manuscript. This project was carried out within the research programme of the Kluyver Centre for Genomics of Industrial Fermentation, which is part of The Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.
Conflict of interest
The authors declare that they have no competing interests.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
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