The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30 Limón et al. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 (24 May 2011) RESEARCH Open Access The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30 M Carmen Limón 1,2* , Tiina Pakula 1 , Markku Saloheimo 1 and Merja Penttilä 1 Abstract Background: Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results: We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1 + Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1 + Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1.Δpgi1 mutants were also affected in the extracellular b-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions: The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1. Background Trichoderma reesei,ananamorphofthespeciesHypocrea jecorina, stands out for its ability to degrade cellulose and hemicellulose. Cellulose is a long polymer of b- 1,4-linked D-glucospyranose units. Degradation of cellulose to glucose requires the synergistic action of different types of enzymes. In T. reesei, at least several endoglucanases (EGI/Cel7B, EGII/Cel5A, EGIII/Cel12A, EGIV/Cel61A, and EGV/Cel45A), exoglucanases (the cellobiohydrolases CBHI/Cel7A and CBHII/Cel6A) and b-glucosidases (BGLI/Cel3A and BGLII/Cel1A) contribute to the total cellulase activity [1,2]. An additional protein, swollenin, disrupts crystalline cellulose structures, making polysaccharides more accessible [3]. Cellulase induction in T. reesei is modulated by several physiological and environmental conditions, such as growth rate [4], sulphur [5] and light [6,7]. Cellulose and disaccharides such as lactose, cellobiose and sophorose (glycosyl b-1,2-glucose) [8,9] can act as inducers * Correspondence: [email protected] 1 VTT, P.O. Box 1000, (Tietotie 2, Espoo), FIN-02044 VTT, Finland Full list of author information is available at the end of the article Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 © 2011 Limón et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and, in fact, lactose is used in industry as a cheap inducer. In T. reesei, lactose is hydrolysed, mainly by an extracellular b-galactosidase, Bga1, to the monosaccharides D-glucose and D-galactose which are taken up by appropriate permeases [10]. Cellulase induction by lactose requires the b-anomer of D-galactose, that cannot be catabolized via the Leloir pathway but can be converted to fructose by an alternative pathway [11]. D- xylose reductase (encoded by xyl1) is the enzyme that catalyzes the first step in the alternative pathway [12]. XYR1 (xylanase regulator 1) is essential for the activation of xyl1. This regulator is also an indispensable transcription factor for expression of hydrolytic enzymes on lactose [13,14]. The majority of the cellulase genes are repressed by the presence of glucose [15,16]. This repression is mediated by CRE1, a two-zinc finger protein that binds to DNA [17,18]. Other negatively- and positively-acting transcription factors have been characterised for cellulase and hemicellulase genes, and their functions have been described in detail [15,19-22]. Cellulases have been used for different applications in food, textile and paper industry and recently have gained interest for the production of bioethanol [19]. Demands of biofuel obtained from renewable feedstock require high amounts of cellulases [23]. One of the best cellulase producer strains is T. reesei Rut-C30. This strain was obtained from the natural isolate QM6a through three mutagenesis steps: (i) UV mutagenesis and screen for the ability to hydrolyse cellulose in repressing conditions; (ii) N-nitrosoguanidine mutagenesis and a similar screen; and (iii) UV mutagenesis, screen for cellulase activity and selection for 2- deoxyglucose resistance to eliminate catabolite repression [24]. The comparison between the genomes of Rut- C30 and QM6a revealed a surprisingly high number of mutagenic events [25,26]. Some of them had been previously characterized, including a truncation of the cre1 gene, encoding the CRE1 repressor; a frameshift mutation in the glucosidase II alpha subunit gene gls2a, involved in protein glycosylation [27]; and an 85-kb deletion that eliminated 29 genes, including transporters, transcription factors, and primary metabolic enzymes [28]. The truncated form of cre1 (allele cre1-1) encodes a truncated protein of only 95 amino acids with a single zinc finger [17]. This mutation, which is equivalent to a null mutation [29], produces catabolite derepression and is one of the main factors responsible for the hypercellulolytic phenotype of Rut-C30. T. reesei is not only biotechnologically important for the cellulase production but also for being an efficient host for large scale production of other enzymes [30]. Different strategies have been applied to improve protein production by this fungus, including the use of strong promoters [31], induction of the unfolded-protein response [32], generation of fusions to a hydrophobic tag [33], control of the specific growth rate [4], and modification of carbon repression [29]. Although Rut-C30 is a good cellulase-producer microorganism, some improvements are still possible [34]. We sought to explore possibilities to engineer the cellulase production capacity of the overproducer Rut-C30 by alteration of a metabolic pathway. The idea was to eliminate phosphoglucose isomerase (PGI) activity in order to redirect the carbon flux through the pentose phosphate pathway (PPP). The two central pathways of carbon metabolism are glycolysis and the PPP (Figure 1). The PPP is the major source of NADPH, needed for the biosynthesis of many biomolecules, in particular fats [35]. It also provides intermediates for the synthesis of amino acids: histidine is synthesised from ribose 5-phos- phate (ribose-5-P), and erythrose 4-phosphate is one of the metabolic precursors needed for the synthesis of aromatic amino acids: phenylalanine, tyrosine and tryptophan [35]. For this reason, it is obvious that the PPP plays an important role in the production of proteins and therefore this pathway is expected to be relevant especially in an organism with an efficient protein production system. The PPP intermediate ribose-5-P is also essential for the synthesis of nucleotides and nucleic acids. PGI catalyzes the second step of glycolysis converting glucose 6-phosphate (glucose-6-P) into fructose 6-phos- phate (fructose-6-P). Since this enzyme is located at the first junction between glycolysis and the PPP, a consequence of the inactivation of PGI could be the rerouting of the metabolic flux through the PPP, which, in turn, would lead to an increase in NADPH production. This is, in fact, the result of the disruption of the pgi gene in the bacterial species Escherichia coli [36]. A similar result was obtained with the pgi mutant of the yeast Kluyveromyces lactis (rag2 mutant), which was able to grow on glucose, bypassing the glycolytic step. This suggests that glucose-6-P was metabolized via the PPP in K. lactis [37]. However, fungal species show differences in thecapacitytousethePPP,and,incontrastwiththe situation in K. lactis and E. coli, the deletion of PGI1 in Saccharomyces cerevisiae prevents growth on glucose [38,39]. The S. cerevisiae PGI1 mutants can grow with fructose as main carbon source, but trace amounts of glucose are required [38,39] because it is the only way to provide glucose-6-P that is required for biosynthesis of many cell components. Boles et al. [40] proposed that the growth defect of the PGI1 mutants of S. cerevisiae on glucose medium was due to a rapid depletion of cytoplasmic NADP, which is needed as a cofactor in the PPP. This defect could be solved either by adding oxidizing agents to restore NADP from NADPH or by Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 2 of 21 over-expressing the NADH-dependent glutamate dehydrogenase, resulting in substrate cycling and coupled conversionofNADtoNADHandNADPHtoNADP [40]. At the same time, high concentrations of glucose prevent growth, probably due either to the toxic effect of high levels of glucose-6-P or to ATP depletion [41]. In addition, there is a great intraspecies diversity in the PPP capacity among S. cerevisiae strains and a low capacity may be the main factor limiting glucose oxidation through this pathway [42]. In this work, we have constructed pgi1 deletion mutants (Δpgi1)inT. reesei Rut-C30 to block the early part of the glycolytic pathway and we have analysed if this mutation leads to the redirection of carbon flux through the PPP. We report the effects of this genetic alteration on morphology, growth, carbon utilisation, lactose metabolism, and, especially, cellulase production. The analysis has been carried out, both in the original cre1-1 background and in a cre1 + background. Results Cloning and analysis of the pgi1 gene of T. reesei To generate a cassette to disrupt the pgi1 gene of T. reesei, it was necessary to clone 5’and 3’fragments of the genomic sequence of the gene. Therefore, the pgi1 cDNA already available was used as a probe to screen a genomic library of T. reesei and the complete genomic sequence was isolated (see Methods). Southern hybridization under low-stringency conditions indicated that the strain Rut-C30 has a single copy of the pgi1 gene (data not shown). In the public genome sequence of T. reesei (Joint Genome Institute, USA) the pgi1 gene is shown as fgenesh1_pm.C_scaffold_29000020, and no other homologous ORFs are found in the Trichoderma reesei database v2.0 [43]. Comparison of the genomic and the cDNA sequences showed that this gene consists of 4 exons and 3 introns. The 554 amino acids of the predicted protein product, PGI (CAG38420), were used in a BLAST search [44]. Figure 1 Glycolysis and the pentose phosphate pathway.Enzymeabbreviations:GPDH,glucose-6-P dehydrogenase and PGI, phosphoglucose isomerase. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 3 of 21 The best scores of overall amino acid identity were 87% with Nectria haematococca PGI (XP_003050252), 86% with PGI from Gibberella zeae PH-1 (XP_386019), 85% with Verticillium albo-atrum (XP_003009491.1), 84% with Glomerella graminicola (EFQ34773.1), 82% with Magnaporthe oryzae (XP_001408812.1), 82% with an unnamed protein of Sordaria macrospore (CBI53771.1), 82% with Neurospora crassa PGI (XP_962135), 80% with Chaetomium globosum (XP_001223630.1), 80% with an unnamed protein product of Podospora anserina (CAP65411.1), and 76% with Aspergillus fumigatus PGI (XP_755312.1). Construction of T. reesei pgi1 deletion strains A disruption cassette, containing a gene conferring resistance to hygromycin flanked by pgi1 promoter and terminator sequences, was generated and used to obtain the Δpgi1 mutants as described in the Methods section (Figure 2A). After transformation of T. reesei Rut-C30 protoplasts, 29 out of 87 resistant colonies showed poor growth and were selected as putative Δpgi1 mutants. The candidates were subjected to 3 rounds of purification on minimal medium (MM) Petri dishes containing 20 g l -1 fructose and 1 g l -1 glucose (and Triton X-100) where they grew as small colonies. The transformants grew as well as the parental strain, however, in liquid rich PD medium with 20 g l -1 fructose (data not shown). DNA isolated from the parental strain and 19 transformants were analysed by Southern blot to verify single copy insertion of the cassette at the pgi1 locus and correct deletion of the pgi1 coding region. Hybridization with a probe specific for the hygromycin resistance gene revealed only a band, indicating a single integration of the deletion cassette in the transformants (results not shown). Hybridization with a pgi1 specific probe (PstI probe), containing part of the pgi1 promoter as well as the 5’end of the coding region of the gene, showed that there were different kinds of transformants (Figure 2B): (i) transformants PD37 and PD51 had the same pattern than the parental strain; (ii) transformants P16 and P39 showed two hybridizing signals, one of 0.6 kb, corresponding to theendogenouscopyofthegene,anda1.9-kbband corresponding to the expected size of the fragment in the deletion cassette; (iii) transformants PD56 and PD57 gave3bands;(iv)finally,13ofthetransformantsgave only a signal corresponding to the disrupted gene (1.9 kb), indicating correct replacement of the gene. Lack of the original coding sequence in the last 13 candidates was confirmed by Southern blot analysis using a pgi1 cDNA fragment as a probe (SacI probe). No band was obtained using this probe (data not shown). These candidates were considered as correct pgi1 deletion mutants, and further analyses were carried out with two of them, P40 and P58. The deletion was functionally confirmed because Δpgi1 strains did not reveal any PGI activity in cell extracts, whereas in the parental strain the PGI activity was 3.498 ± 0.026 nkat mg -1 protein. The activity was measured twice from mycelia grown in MM with glucose. Introduction of the cre1 wild-type allele (cre1 + )inaΔpgi1 mutant As explained in the Background section, Rut-C30, the parental strain of our Δpgi1 mutants, has a deletion in the cre1 gene (allele cre1-1), which is an important factor to explain its hypercellulolytic phenotype. Presumably, this alteration in cre1 could have an impact in the phenotypes that we intended to study in the pgi1 mutants. In order to get a control for comparison and better evaluation of the effects of the pgi1 deletion, we generated a version of one of the mutants carrying a wild-type copy of cre1, allele cre1 + . A 6-kb genomic fragment containing the gene cre1 with its own promoter was introduced in the Δpgi1 mutant P40 (Figure 3A). 17 putative transformants were selected in media with acetamide, fructose and glucose. Genomic DNA was isolated from QM6a, P40 and the putative cre1 + Δpgi1 transformants. The DNA was digested with PstI and analysed by Southern blot using a 1.2-kb DIG-labelled probe corresponding to the cre1 ORF. A PstI 661-bp fragment of cre1 was not present in the allele cre1-1. Transformants with the inserted cre1 gene were identified because they showed this fragment when hybridized with a 868-bp probe specific for the region that is absent in the allele cre1-1 (data not shown). Four of these transformants, CP3, CP5, CP26 and CP28, were further analysed by Southern blot hybridisation. Genomic DNA of strains QM6a, P40 and the four selected cre1 + Δpgi1 mutants were digested with EcoRV, SacIandXbaI and hybridized with the 868-bp probe. This analysis showed that CP3, CP5 and CP26 contained only one insertion of wild-type cre1 while CP28 contained two copies of the gene (Figure 3B). Mutants CP26 and CP28 were chosen for further analysis. Morphology of the Δpgi1 and the cre1 + Δpgi1 strains First, we studied the growth and morphology of the pgi1 mutants in liquid and solid media. The Δpgi1 and cre1 + Δpgi1 mutants were able to grow in liquid cultures in MM with glucose. However, differences in morphology were observed. Macroscopically, a pellet-like growth was apparent for the mutants but it was not observed for the parental strain (Figure 4). Under a microscope, the hyphae of the mutant strains were wider and more branched compared to the parental strain, and contained balloon-like structures (Figure 5A and 5B). In Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 4 of 21 liquid cultures containing fructose as a sole carbon source, growth of the Δpgi1 strains ceased at early stages soon after germination. On the other hand, the parental strain showed normal phenotype with long hyphae (Figure 5C) whereas the germinated hyphae of the Δpgi1 and cre1 + Δpgi1 strains remained very short in fructose (Figure 5D and data not shown). Mutants also produced lower amounts of spores when grown on potato dextrose agar (PDA) with 20 g l -1 fructose plates (data not shown). Under the microscope such spores showed dark organelles and some of them were swollen (Figure 5F) in comparison to those produced by RutC-30 (Figure 5E). The growth of Δpgi1 and cre1 + Δpgi1 mutants was measured on Petri dishes with different carbon sources. On glucose as the sole carbon source, Δpgi1 mutants started to grow later than in the other conditions measured (Figure 6A). Their growth on media supplemented with any of the other carbon sources along with glucose or glucose as sole carbon source was always slower than the growth of the parental strain. The Δpgi1 mutants grewinmediawithfructoseandglucose(Figure6B), Figure 2 Map and Southern analysis of Rut-C30 and putative Δpgi1 mutants.(A) Rut-C30 genomic pgi1.(B) Correct insertion of the deletion cassette in the Δpgi1 mutants. Probes used for Southern experiments are indicated as bars that are located in the corresponding region of the DNA where they hybridize. (C)Southern analysis. Genomic DNA of strain RutC-30 and 19 putative transformants were digested with PstI. Filter was probed with a 0.6-kb PstI fragment comprising part of the coding region and part of the 5’UTR of the pgi gene. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 5 of 21 with galactose and fructose (Figure 6C), and with lactose and fructose (Figure 6D). When grown on plates of medium with fructose and glucose, the Δpgi1 mutants did not show aerial hyphae and, compared to the parental strain, the mutant colony morphology was thinner at the colony edges. The diameters of the Δpgi1 colonies were larger (Figure 6C) and their mycelia were thicker (Figure 7) on plates with galactose and fructose than on the other sugar combinations tested. Colony shape of Δpgi1 on lactose and fructose was more symmetrical than in any of the other combinations tested. Δpgi1 mutants also grew in media with glycerol and glucose although mycelia of all the strains tested, including Rut- C30, were less compact (data not shown). No growth was observed on MM plates with xylose and glucose (Figure 7). When galactose or lactose was used as the sole carbon source only a weak growth was observed after 13 days (Figure 7). The radial growth of the cre1 Figure 3 Southern blot analysis of cre1 + Δpgi1 mutants.(A) Map of genomic cre1 gene. Arrows indicate fragments released with XbaI and EcoRI that hybridize with the probe (in red). Southern analysis of strains QM6a, P40, CP3, CP5, CP26 and CP28: (B) Genomic DNA was digested with XbaI. (C) Genomic DNA was digested with EcoRV. M1 = DNA Molecular Weight Marker II, digoxigenin-labeled. M2 = DNA Molecular Weight Marker II, digoxigenin-labeled. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 6 of 21 + Δpgi1 mutants was comparable to the radial growth of the Δpgi1 mutants in most of the sugar combinations (Figure 6 E, F, G, H and Figure 7) and they did not grow on either xylose or xylose and glucose (Figure 7). The conclusion of this section is that carbon sources that feed to the metabolism after the PGI step in glycolysis, i.e., fructose or glycerol, did not support growth of the mutants, but growth was restored if glucose was added to the media (Figure 6). Moreover, Δpgi1 did not grow on other sugars such as, lactose or galactose, unless fructose was also added. In summary, for the conditions tested, glucose was essential for the growth of the mutants but was not enough for normal growth. In addition, the effects of the pgi1 mutation on growth and morphology were independent of the cre1-1 mutation. Cellulase activity Next, we investigated the impact of the pgi1 mutations on cellulase production of T. reesei.TheΔpgi1 mutants produced higher total cellulase activity than Rut-C30 in MM with glucose (Figure 8A). The maximum levels for P40andP58were0.46nkatml -1 and 0.27 nkat ml -1 , respectively, and the maximum level for the parental strain was 0.21 nkat ml -1 . The beginning of cellulase production in the cultures of the mutants coincided with the beginning of glucose consumption and the production ceased when the biomass reached the maximum level in the cultures. However, in Rut-C30 cellulase activity started to be detected much later, after 235 h of culture. The mutants also produced higher cellulase activity than Rut-C30 in MM with glucose and fructose (0.56 nkat ml -1 and 0.28 nkat ml -1 in the pgi1 mutants vs 0.04 nkat ml -1 in Rut-C30) and the activity level did not decrease during the cultivation (Figure 8B). Cellulase production in the mutants started slightly earlier in MM with fructose and glucose than in MM with only glucose but maximal production was similar in both media. In contrast, Rut-C30 had almost undetectable activity in this medium. In conclusion, the cellulase production by the Δpgi1 mutants exceeded the derepressed level of cellulase production in the parental strain. Figure 4 Pellet-like growth of the mutants in liquid cultures. Samples from the cultures and cultures of Rut-C30, P40 and CP28 that are below. Strains were grown in MM with glucose and fructose at 30°C and 150 rpm. Photos were taken after 3 days of growth. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 7 of 21 Lactose is commonly used as an inducing carbon source for cellulase production and, consistent with that, higher cellulase activity levels were obtained in the presence of lactose. Rut-C30 produced 2-fold more activity than P40 mutant and 3-fold more than P58 mutant in this medium (Figure 8C). Rut-C30 reached 1.9 nkat ml -1 of total cellulase activity, whereas maximal cellulase activities for P40 and P58 were 1 nakt ml -1 and 0.6 nkat ml -1 , respectively. Cellulase activity started to be detected in the three strains after 6 days of growth and increased with time. All the strains tested exhibited increased cellulase activity in MM with lactose and fructose, in comparison to medium with lactose and glucose. However, whereas in Rut-C30 a 10-fold increase was observed, in the Δpgi1 strains the activity increased about 4 to 5-fold (Figure 8D). Cellulase activity of Rut-C30 in MM with lactose and fructose was detected earlier than in MM with lactose and glucose. After reaching a maximum of 18 nkat ml -1 at 142 h, there was a steep decline to 2.5 nkat ml -1 at 236 h. This correlated with a decrease in extracellular protein concentration (Additional file 1B). Production was kept up until the end of the cultivation. However, in MM with lactose and glucose the decrease in activity occurred much later, after 280 h (Figure 8C). Δpgi1 mutants produced a maximum of 2-5 nkat ml -1 after 315 Figure 5 Comparison of morphology of T. reesei Rut-C30 and a Δpgi1 mutant.(A) Rut-C30 mycelium grown in MM with glucose. (B) Δpgi1 mycelium grown in MM and glucose. (C) Rut-C30 mycelium grown in MM and fructose. (D) Δpgi1 mycelium grown in MM with fructose. Mycelia were grown for 119 h in 28°C at 200 rpm. (E) Conidia from Rut-C30 mycelia grown on PDA with fructose. (F) Conidia from a Δpgi1 grown on PDA with fructose. Scale bars represent 10 μm. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 8 of 21 C30,whengrowninMMwithglucose.Inthese mutants, glucose should be metabolized by the PPP instead of by glycolysis. However, glucose-6-P could be accumulated in the cells for two reasons: (i) the PGI step is blocked and glucose-6-P cannot be converted to fructose-6-P and (ii) the flux through the PPP is too slow. E. coli produces a glucose transporter that is downregulated upon accumulation of a high concentration of hexose-phosphate [55]. This downregulation modulates glucose uptake to avoid over accumulation of glucose-6-P and fructose-6-P, which are potentially toxic to the cell [56,57]. One possible explanation for the reduction of glucose consumption rate in the Trichoderma Δpgi1 mutants could be a similar feedback regulation on glucose transporters. If glucose-6-P were metabolized slowly through the PPP in the mutants, glucose-6-Pwouldbeaccumulatedandwouldinhibit expression of glucose transporters. When mutants were grown with a combination of sugars: MM with fructose and glucose or MM with fructose and lactose, their fructose consumption was faster than that of Rut-C30. Fructose must be rapidly transported and metabolized through glycolysis. As previously discussed, the slow consumption of lactose by the pgi1 mutants and the differences detected in lactose concentration in the cultures of the different strains, could be explained by differences in b-galactosi- dase activities. Gene expression gpdh mRNA levels did no increase in the mutants growing on glucose (Figure 9A), a result that correlated with the GPDH activity measurements (data not shown). This result suggests that glucose was not inducing gpdh. However, it cannot be discarded that the PPP fluxes were not regulated at gpdh level. The relative expression of the cellulase activator gene, xyr1, is higher in the Δpgi1 mutants after the transfer to glucose-containing media, which could help to explain the differences in cellulase activity under conditions of catabolite repression. The almost undetected activity in Rut-C30 cannot be explained by the mRNA levels of the activator. However, after several hours of incubation in media with glucose, Rut-C30 and the Δpgi1 mutants did not show significant differences of xyr1 expression, except for a transientincreaseinP40after6hofincubationinglucose (Figure 9B). Therefore, as already mentioned in the Results section, the differences observed in cellulase activities between Rut-C30 and P40 could not be totally explained by the expression of the regulator xyr1. In lactose, xyr1 relative expression was not statistically different between P40 and Rut-C30, but the expression in P58 was significantly lower (P< 0.001). In Rut-C30 and P40 the xyr1 expression increased with time on lactose, as described for a Δcre1 mutant and for Rut- C30 [58]. The amount of xyr1 mRNA in the strain P58 started to decrease after 6 to 14 hours of incubation in media with lactose. There were no significant differences in the expression of xyl1 when the different strains were grown in media with glucose (Figure 9C). However, on lactose the xyl1 expression was lower in the Δpgi1 mutants than in Rut- C30. The differences were statistically significant for P40 and P58 at 14 h after the transfer (P< 0.001). This result was unexpected because XYR1 regulates the expression of xyl1 on lactose [14]. In this case, there should be other factors affecting the expression of xyl1. The differences in b-galactosidase activity found between Rut-C30 and the Δpgi1 mutants agree with the accumulation of xyl1 mRNA in the strains that have beenstudied.TheenzymeD-xylosereductase,XYL1, converts D-galactose into galactitol [12]. It was formerly shown that galactitol induces b-galactosidase [14,59]. Our results are in agreement with previous data, because the mutant P58, that exhibited a reduced expression of xyl1, had lower b-galactosidase activity. Differences between Δpgi1 mutants P40 and P58 The Δpgi1 strains analysed in this work, P40 and P58, exhibited some differences in the parameters that were measured. For instance, they differed in cellulase production, consumption of glucose and other sugars, patterns of growth, andexpressionofxyr1 and xyl1 under certain conditions. Although the deletion of pgi1 was the same in both mutants, according to Southern analysis,thephenotypicdifferencesareindicativeofsome additional genotypic alteration. This is not surprising if we take into account the poor growth of the mutants in the selective medium. A consequence could be the accumulation of spontaneous suppressor mutations that could partially alleviate the growth defect. This was the case of pgi1 suppressor mutants isolated in S. cerevisiae that enhanced glucose catabolism through the PPP [40,60,61]. A suppressor gene was GDH2 coding for the NAD-dependent glutamate dehydrogenase [40]. The possibility of suppressor mutations made advisable the analysis of more than one independent mutant to get general conclusions about the effects of the pgi1 deletion. In fact, the behaviour of both mutants was similar, although P40 showed more differences than P58, when compared with the parental strain, for certain parameters. Interestingly, the main conclusions of this work (i.e., growth phenotypes, cellulase production, sugar consumption) were confirmed in both mutants. Conclusions We have deleted the pgi1 gene in the hypercellulolytic T. reesei mutant Rut-C30. The resulting strains were Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 15 of 21 able to grow using glucose as the only carbon source, suggesting that the PPP is active in T. reseei.These Δpgi1 mutants exhibited higher total cellulase activity under repressing conditions in comparison to Rut-C30. The increase in activity is partially explained by the pgi1 mutation,andpartiallybytothegeneticinteraction between the pgi1 mutation and the cre1-1 mutation that was already present in RutC-30. In contrast, the morphological and growth alterations observed in the mutants may be attributed to the pgi1 mutation since similar effects were observed in cre1 + and cre1-1 backgrounds. In summary, since the pgi1 mutation produces an increase in cellulase activity in certain media, even though not under inducing conditions, it could be interesting to use this mutation, in combination with others, to improve the hypercellulolytic phenotypes of T. reesei strains. Overexpression of bga1 gene could be explored as a way to increase induction by lactose in pgi1 mutants. Another strategy of improvement could be the co-cultivation of Δpgi1 mutants with overproducer strains under inducing conditions, taking advantage of their different sugar preferences and their different production profile. Methods Strains T. reesei strain Rut-C30 (ATCC 56765, VTTD-86271) [62] was used for gene disruption, strain VTT-D-80133 [63] was used for the preparation of a genomic cosmid library and the natural isolate QM6a (ATCC13631, VTT-D- 071262T) was used as a control. Escherichia coli strains JS4 and DH5awere used for library and plasmid constructions, respectively, while strain TOP10F’wasusedasa host for cloning PCR products in the TOPO vector. Media and culture conditions Conidia of T. reesei Rut-C30, pgi1 and cre1 + Δpgi1 mutants for inoculating liquid cultures were collected from mycelia grown for 7 days at 28°C on potato dextrose agar (PDA) and PDA supplemented with 20 g l -1 fructose, respectively. Unless otherwise stated, the liquid cultures were grown on minimal medium (MM) [8] with 10 g l -1 of each carbon source added (either glucose, fructose, lactose or a combination of them). Conidia (4 × 10 7 )ofRut-C30orΔpgi1 strains were inoculated in 400 ml of medium in 2-liter flasks and incubated on a rotary shaker at 200 rpm at 28°C. For determination of dry weight, two sample aliquots were weighed and collected on pre-weighed filter disks (Whatman GF/B 55 mm Ø). The biomass was washed with approximately 50 ml of demineralised water and dried overnight in an oven at 105°C, and afterwards the disks were weighed again. Triplicates of each strain were cultured for each condition. For comparison of cellulase activity among all the strains, Rut-C30, QM6a, Δpgi1 and cre1 + Δpgi1 mutants were cultured in 500-ml flasks containing 100 ml of MM with glucose and fructose or MM with lactose and fructose. Mycelia were incubated for 7 days at 30°C in a rotary shaker at 150 rpm. b-galactosidase activity was measured from these culture supernatants. For gene expression experiments, strains were first grown in 500-ml flasks containing 100 ml of MM with glucose, fructose and 2 g l -1 peptone at 150 rpm and 30° C. After 30 h of culture, mycelia from each strain were filtered, washed and equal amounts of each strain were resuspended in MM containing glucose or lactose. Samples were immediately taken after the transfer (t = 0) and after 6 and 14 h of culture. Two independent experiments were carried out. To determine hyphal growth on agar plates, a 7-mm plug was inoculated in the center of each 8-cm Petri dish containing MM with the following combination of sugars: glucose, fructose, fructose and glucose, galactose, galactose and fructose, glycerol, glycerol and glucose, lactose, lactose and fructose, xylose, xylose and glucose. Plates were incubated at 22°C for 14 days and colony diameter was measured daily. The data shown are the average of three independent experiments. Error bars indicate standard deviation. Bacterial host strains were grown in LB medium at 37°C. Isolation of the chromosomal pgi gene The chromosomal pgi1 genewasisolatedfromagenomic cosmid library of T. reesei (strain VTT-D-80133) in the cosmid p3030 (Hohn & Hinnen, unpublished). The library was screened using a pgi1 cDNA fragment as a probe. All positive clones corresponded to the same cosmid. DNA from cosmid K4.1 was digested with enzymes StuI, PstIorSacI, blotted and hybridized with a fragment of the pgi1 cDNA. Positive fragments of 2.5 and 5 kb from the StuI digestion and the positive 5-kb fragment from the PstI digestion, were subcloned in pBluescriptSK+ (Stratagene, La Jolla, USA). The resulting plasmids were used to clone the gene. ABI PRISM Bigdye™terminator sequencing kit (Australia) was used to check plasmid constructions and for sequencing the chromosomal pgi1 gene. Deletion of gene pgi1 from the Trichoderma genome The primers used for PCR amplification of pgi1 promoter (2.1 kb fragment) from the cosmid clone were Oligo-KpnI-PacI-Prom (+) 5’-CCG AGA GGT ACCTTA ATT AAG GCC CCA TCC GTT CTT CCA TGA C-3’,whereKpnI restriction site is underlined and PacI site is in bold, and Oligo-PGI-KpnI (-) 5’-CGG GGC ATT GGT ACC GTT GGA GAG GTT GTG GTT GAA GTA-3’,whereKpnI restriction site is Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 16 of 21 underlined. The PCR reaction mixture contained 0.1 U μl -1 Advantage polymerase (BD Biosciences Clontech), 0.2mMdNTP,1.1mMMg(CH 3 COO) 2 ,10μM of each primer and 20 ng of the cosmid DNA in a 50 μlfinal volume. The PCR program used was: 32 cycles of 94°C 25 s, 50°C 3 min, 72°C 3 min, followed by a single cycle of 67°C 7 min. The amplified fragment was isolated from a 10 g l -1 agarose gel, purified through Qiaquick Spin Columns (Qiagen, Netherlands), ligated to TOPO TA cloning vector (Invitrogen) and transformed to TOP10 E. coli competent cells. The fragment was isolated from the TOPO vector by digesting with Asp718 (isoschizomer of KpnI) (Roche Molecular Biochemicals) and cloned in vector pARO21 (5.7 kb) [64], previously digested with Asp718, to obtain plasmid pCL23. The pgi1 terminator was amplified by PCR from the cosmid clone as a 1.6-kb fragment using primers Term1.6For- ward, 5’-CTGGGGCGTCGAAGCTTGGGCAAGGT- CCTT-3’(Restriction site HindIII underlined), and Term1.6Reverse, 5’-GGCAAGCTTAATTAAGGC AGGGCGAGCTGAACAA-3’(restriction site HindIII is underlined and restriction site PacI is in bold). The conditions for the PCR reaction were as described above. This 1.6-kb fragment contained 0.3 kb of the 3’end of the coding region of the pgi1 gene and 1.3 kb of the pgi1 terminator. The amplified fragment was digested with HindIII and ligated to pCL23, which contained the pgi1 promoter and the hygromycin resistance marker to generate plasmid pCL28 (9.5 kb). A fragment of 6.7 kb containing the cassette for deletion was released from pCL28 digesting with PacIandusedfortransformation of T. reesei. Transformation procedures E. coli TOP10F’was transformed by electroporation according to the manufacturer’s instructions (Bio-Rad). T. reesei protoplasts were transformed with 5.5 μgof the pgi1 deletion cassette as described earlier [65]. The transformed protoplasts were plated on MM medium where glucose was replaced by 20 g l -1 fructose and 1 g l -1 glucose, and hygromycin (150 μgml -1 ) was added for selection of the transformants. Monosporic cultures from transformant strains were obtained on selection media (MM medium with 20 g l -1 fructose, 1 g l -1 glucose, 150 μgml -1 hygromycin and 1 ml l -1 Triton X- 100). Introduction of gene cre1 in a Δpgi1 background was done by cotransformation with a 6-kb SalI-HindIII genomic fragment from plasmid pMI-41 [17], containing cre1, together with plasmid pTOC202. Strains were selected for growth on acetamide in MM with 20 g l -1 fructose, 5 g l -1 glucose and 10 mM acetamide. The putative transformants were submitted to three selection rounds on selection media to purify them. Nucleic acid hybridizations Genomic DNA of T. reesei strains was isolated using Easy DNA™kit (Invitrogen, CA). Southern blot hybridizations were carried out using standard methods [66]. Probes of gene pgi1 for Southern blot analyses were a 1.4 kb fragment of pgi1 cDNA obtained from plasmid pMS57 after digestion with SacI, and a 0.6 kb fragment obtained from plasmid pCL5 after digestion with PstI, the latter containing a fragment of the pgi1 promoter and the coding region. For detection of the hygromycin resistance gene, either a NsiI-XbaI fragment from the plasmid pRLMex30 [20] or a 782-bp XbaI-SacII fragment of the hygromycin gene, obtained from plasmid pARO21 [64], were used as probes. The restriction enzymes used for digesting genomic DNA for Southern blot analyses were BstXI, EcoRI and NdeI. Genomic DNA of wild type QM6a, P40, and 17 putative cre1 + Δpgi1 transformants was digested with PstIto discard transformants without integration of cre1. To test the number of integrations of the cre1 cassette, genomic DNA from strains QM6a, P40, CP5, CP26 and CP28 was digested with EcoRV, SacIandXbaI. A 1.2-kb probe corresponding to the complete open reading frame (ORF) was obtained from plasmid pMI-41 with primers 1592: 5’-GGCGGATCCATGCAACGAGCACAGTCTGCC-3’ and 1590: 5’-GGCGGATCCCTACATGGCATCCAT- GAGGTC-3’.Aspecificprobeforaprotein-coding region, which did not hybridize with the allele cre1-1 of Rut-C30, corresponded to nucleotides +342 to +1209 of cre1. This specific probe was amplified from plasmid pMI-41 and DIG-labelled with the PCR DIG probe synthesis kit using primers 1842: 5’-CGCGGATC- CAGGCGGGGCAACAAGGGCAG-3’, and 1844: 5’- GTGGGAATTCCTACATCCGATCCATGAGGTCGCC-3’. The Southern detection was carried out with detection starter kit II using the standard procedure (Roche Diagnostics GmbH, Mannheim, Germany). RNA isolation and expression MyceliawerelysedinaFast-Prep ® -24 homogenizer (MP™Biomedicals LLC Europe, France) using zirconia microbeads by 2 pulses of 30 s at 6 m/s. Total RNA was isolated using the RNeasy Plant Mini kit (Qiagen, Spain). 250 ng of RNA samples were treated with 1 U of rDNAsa I (USB, Affymetrix, Inc) for 15 min at 25°C and the reactions were stopped with 1 μlof50mM EDTA and incubated at 65°C for 10 min. Total RNA concentration was estimated using a Nanodrop ND- 1000 spectrophotometer (NanoDrop Technologies, Wilmintong, DE, USA). RT-PCR mixtures contained 1 × Power SYBR-Green PCR Master Mix (Applied Biosystems), 0.125 μlMulti- Scribe Reverse Transcriptase (50 U μl -1 ), 0.125 μl RNAse Inhibitor (10 U μl -1 ), 100 ng of total RNA and 8 Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 17 of 21 μM of each primer, in a total volume of 25 μl. RT-PCRs were performed in an ABI 7500 (Applied Biosystems). The PCR program consisted of 30 min at 48°C for retrotranscription, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. Dissociation curves were done to test amplification validity. Primer sets to analyse gene expression by RT-PCR were: gpdhF:5’-GGGCGG- CTACTTTGATGAGTT-3’and gpdhR:5’-TGGTTCTG- CATGACGTCTCG-3’for glucose-6-P dehydrogenase gene; xyrF:5’-GAGCTTTCGAGTTCACGCATG-3’and xyrR:5’-CCCAGCAGTACCCGTTGAATT-3’for the xylanase regulator 1, xyr1 (AF479644) [21]; xyl1F:5’- CTGTGACTATGGCAACGAAAAGGAG-3’and xyl1R: 5’-CACAGCTTGGACACGATGAAGAG-3’[14] for xy1l, the gene for D-xylose reductase; actF:5’-TGA- CATGGCTGGTCGTGATC-3’and actR:5’-ATGT- CACGGACGATTTCCCTC-3’for actin-encoding gene (X75421), used as an endogenous control. Primer design and relative gene expression were done as described by Rodríguez-Ortiz et al. [67]. Each RTPCR analysis was achieved in duplicate to confirm repetitiveness. The values given are the mean of 2 independent experiments with 2 measurements each one. Error bars indicate standard deviation. Preparation of cell extracts Mycelial samples were harvested by filtration through Whatman GF/B glass fiber filters, washed with 7 g l -1 NaCl,frozeninliquidnitrogenandstoredat-80°C.Cell extracts from frozen mycelium were prepared as described by Pakula et al.[68].Briefly,equalamountsof mycelia were resuspended in 100 mM Tris buffer pH 7.4, containing 20% glycerol, and 1 tablet of Complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, USA) per 50 ml of buffer. Mycelia were disrupted by 20 cycles of sonication for 8 s followed by 30 s on ice between the cycles and centrifuged at 14,000 × gfor 10 min. The homogenate was used for enzyme assays. Protein concentrations of the cell extracts were measured using Bradford method (Bio-Rad, Hercules, CA, USA) using bovine serum globulin (500-0007, Bio-Rad, Hercules, CA, USA) as a standard protein. Enzyme assays Phosphoglucose isomerase (EC 5.3.1.9) was assayed from cell extracts as described by Maitra and Lobo [69]. All enzyme assays were carried out in a Cobas Miras automated analyser (Roche) at 30°C. Phosphoglucose isomerase (PGI) activity was measured by adding the cell extract to a reaction mixture containing 10 mM sodium phosphate buffer (pH 7.0), 0.2 mM NADP + ,1μg glucose-6-P dehydrogenase (Boehringer-Mannheim, GmbH, Germany). The reaction was started adding fructose-6-P (Boehringer-Mannheim, GmbH, Germany) to a final concentration of 2 mM. The activity was calculated from the rate of the decrease in NADH absorbance at 340 nm. For cellulase activity, culture aliquots were filtered as described above and filtrates were kept at -20°C until measurement. Total cellulase activity in culture supernatants were measured as described by van Tilbeurg [70]. 4-methylumbelliferyl-b-lactoside (MUL) (Sigma, St. Louis, MO, USA) was used as the substrate (0.17 mM). MUL and culture filtrates were incubated in 50 mM sodium acetate buffer (pH 5.0) with a final volume of 100 μl for 5 min at 37°C. The reaction was stopped with 100 μlof1MNa 2 CO 3 . Individual activities of cellobiohydrolase (CBH) and endoglucanase were estimated by adding 10 mM cellobiose (inhibits mainly CBH) or 100 mM glucose (inhibits b-glucosidases) to the assay, respectively. Fluorescence was measured on a Varioskan microtiter plate reader (Thermo Scientific, USA) at excitation and emission wavelengths of 350 and 450 nm. Measurements were done from three cultures replicates. Extracellular b-galactosidase activity was determined in cultures grown in MM with lactose and fructose for 7 days at 30°C and 150 rpm. Supernatants were filtered and concentrated by ammonium sulphate precipitation [71]. The resulting concentrated samples were dialyzed against 3 liters of 50 mM sodium citrate buffer pH 4.5 at 4°C. 200 μl samples were incubated with 850 μlof50 mM sodium citrate buffer pH 4.8 at 50°C. The reaction was carried out by addition of 150 μlof10mMo-nitro- phenyl-b-D-galactopyranoside as a substrate and incubated for 20-40 min. The reaction was stopped with 500 μlof1MNa 2 CO 3 . Activity was measured at 405 nm against a supernatant blank for each sample in a spectrophotometer (Beckman DU ® ,USA).Oneunitofbgalactosidase activity was defined as the amount of enzyme releasing 1 μmol of p-nitrophenyl per min under the conditions used. Extracellular protein concentration was determined by Bradford method (Bio-Rad, Hercules, CA, USA) using bovine serum globulin (500-0007, Bio-Rad, Hercules, CA, USA) as a standard protein. Measurement of sugar concentrations Fructose and glucose concentrations in culture medium were measured by HPLC (Waters, Milford, Massachusetts, USA) using an Aminex HPX-87H column (Bio Rad Labs, USA) with a 2487 dual aabsorbance detector and 5 mM H 2 SO 4 (Titrisol, Merck) as an eluent at a flow rate of 0.6 ml min -1 at 55°C. Glucose and fructose were used as standards. The data was processed using Water Millenium software. Lactose and galactose concentrations were analysed enzymaticaly using the Boehringer 176 303 Lactose kit (Boehringer Mannheim, Germany) on the Cobas Miras automated analyser (Roche Applied). Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 18 of 21 Microscopy Samples were observed under a microscope (model BHS-RFK, Olympus Optical Ltd., Tokyo, Japan). The microscope was connected to a video camara (Sensicam, PCO CD Imaging, Kelheim, Germany) and digital image analysis software (analySIS ® 3.0, Soft Imaging System GmgH, Münster, Germany). Statistical analysis Statistical significance of differences in gene expression among Trichoderma strains were evaluated with a Student’st-test. Additional material Additional file 1: Extracellular protein in media with lactose. Extracellular protein concentration was measured in the supernatants of cultures of Rut-C30, P40 and P-58 in: (A) MM with lactose and glucose. (B) MM with lactose and fructose. Cultures were grown at 30°C and 200 rpm. Extracellular protein was measured from triplicates of each strain. Errors indicate standard deviation. Abbreviations Fructose-6-P: fructose 6-phosphate; glucose-6-P: glucose 6-phosphate; GPDH: glucose 6-phosphate dehydrogenase; MUL: 4-methylumbelliferyl-β-lactoside; PGI: phosphoglucose isomerase; PMI: phosphomannose isomerase; PPP: pentose phosphate pathway; ribose-5-P: ribose 5-phosphate. Acknowledgements This work was supported by an individual Marie Curie Fellowship (QLK-CT- 1999-51554) and a José Castillejo Mobility Fellowship from the Spanish Government (Ministerio de Educación y Ciencia JC2007-00186) given to MCL, and the research program “VTT Industrial Biotechnology”(Academy of Finland, Finnish Centre of Excellence Program, 2000-2005, Project no. 64330). We thank Aili Grundström for skilled technical assistance, Dr. Marilyn Wiebe for technical advice and Dr. Peter Richard for technical advice and discussion. We also thank Dr. Francisco Ramos-Morales for discussion and critical reading of the manuscript and Dr. Javier Avalos for text revision. Author details 1 VTT, P.O. Box 1000, (Tietotie 2, Espoo), FIN-02044 VTT, Finland. 2 Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Seville, Spain. Authors’contributions MCL designed and performed all the experiments, carried out the data analysis and drafted the manuscript. TP participated in the design of the study, data analysis and drafting of the manuscript. MS participated in the coordination of the study. MP designed the study and helped to draft the manuscript. All authors read and approved the manuscript. Competing interests The authors declare that they have no competing interests. Received: 2 September 2010 Accepted: 24 May 2011 Published: 24 May 2011 References 1. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJ, Yao J, Ward M: Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.J Biol Chem 2003, 278(34):31988-13997. 2. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, et al:Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 2008, 26(5):553-560. 3. Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssönen E, Bhatia A, Ward M, Penttilä M: Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 2002, 269(17):4202-4211. 4. Pakula TM, Salonen K, Uusitalo J, Penttilä M: The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei.Microbiology 2005, 151(Pt 1):135-143. 5. Gremel G, Dorrer M, Schmoll M: Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei). BMC Microbiol 2008, 8:174-191. 6. Schmoll M, Kubicek CP: ooc1, a unique gene expressed only during growth of Hypocrea jecorina (anamorph: Trichoderma reesei) on cellulose. Curr Genet 2005, 48(2):126-133. 7. Seibel C, Gremel G, do Nascimento Silva R, Schuster A, Kubicek CP, Schmoll M: Light-dependent roles of the G-protein αsubunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei). BMC Biol 2009, 7:58. 8. Ilmén M, Saloheimo A, Onnela ML, Penttilä ME: Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.Appl Environ Microbiol 1997, 63(4):1298-1306. 9. Mandels M, Parrish FW, Reese ET: Sophorose as an inducer of cellulase in Trichoderma viride.J Bacteriol 1962, 83:400-408. 10. Seiboth B, Hartl L, Salovuori N, Lanthaler K, Robson GD, Vehmaanperä J, Penttilä ME, Kubicek CP: Role of the bga1-encoded extracellular βgalactosidase of Hypocrea jecorina in cellulase induction by lactose. Appl Environ Microbiol 2005, 71(2):851-857. 11. Fekete E, Seiboth B, Kubicek CP, Szentirmai A, Karaffa L: Lack of aldose 1- epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): a key to cellulase gene expression on lactose. Proc Natl Acad Sci USA 2008, 105(20):7141-7146. 12. Seiboth B, Gamauf C, Pail M, Hartl L, Kubicek CP: The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D- galactose catabolism and necessary for β-galactosidase and cellulase induction by lactose. Mol Microbiol 2007, 66(4):890-900. 13. Stricker AR, Grosstessner-Hain K, Wurleitner E, Mach RL: Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina.Eukaryot Cell 2006, 5(12):2128-2137. 14. Stricker AR, Steiger MG, Mach RL: Xyr1 receives the lactose induction signal and regulates lactose metabolism in Hypocrea jecorina.FEBS Lett 2007, 581(21):3915-3920. 15. Aro N, Pakula T, Penttilä M: Transcriptional regulation of plant cell wall degradation by filamentous fungi. FEMS Microbiol Rev 2005, 29(4):719-739. 16. Kubicek C, Penttilä M: Regulation of production of plant polysaccharide degrading enzymes by Trichoderma.In Trichoderma and Gliocladium. Volume 2. Edited by: Harman GE, Kubicek CP. London: Taylor 1998:49-72. 17. Ilmén M, Thrane C, Penttilä M: The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 1996, 251(4):451-460. 18. Strauss J, Mach RL, Zeilinger S, Hartler G, Stoffler G, Wolschek M, Kubicek CP: Cre1, the carbon catabolite repressor protein from Trichoderma reesei.FEBS Lett 1995, 376(1-2):103-107. 19. Kubicek CP, Mikus M, Schuster A, Schmoll M, Seiboth B: Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina.Biotechnol Biofuels 2009, 2:19-32. 20. Mach RL, Zeilinger S: Regulation of gene expression in industrial fungi Trichoderma.Appl Microbiol Biotechnol 2003, 60(5):515-522. 21. Rauscher R, Wurleitner E, Wacenovsky C, Aro N, Stricker AR, Zeilinger S, Kubicek CP, Penttilä M, Mach RL: Transcriptional regulation of xyn1, encoding xylanase I, in Hypocrea jecorina.Eukaryot Cell 2006, 5(3):447-456. 22. Schmoll M, Kubicek CP: Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol Immunol Hung 2003, 50(2-3):125-145. 23. Alriksson B, Rose SH, van Zyl WH, Sjöde A, Nilvebrant NO, Jönsson LJ: Cellulase production from spent lignocellulose hydrolysates by recombinant Aspergillus niger.Appl Environ Microbiol 2009, 75(8):2366-2374. 24. El-Gogary S, Leite A, Crivellaro O, El-Dorry H, Eveleigh DE: Trichoderma reesei cellulase_from mutants to induction. In Trichoderma reesei cellulases: Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 19 of 21 biochemistry, genetics, physiology and application. Edited by: Kubicek CPE DE, Esterbauer, H, Steiner, W Kubicek-Pranz, EM. Cambridge: Royal Society of Chemistry; 1990:200-211. 25. Le Crom S, Schackwitz W, Pennacchio L, Magnuson JK, Culley DE, Collett JR, Martin J, Druzhinina IS, Mathis H, Monot F, Seiboth B, Cherry B, Rey M, Berka R, Kubicek CP, Baker SE, Margeot A: Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing. Proc Natl Acad Sci USA 2009, 106(38):16151-16156. 26. Vitikainen M, Arvas M, Pakula T, Oja M, Penttilä M, Saloheimo M: Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties. BMC Genomics 2010, 11:441. 27. Geysens S, Pakula T, Uusitalo J, Dewerte I, Penttilä M, Contreras R: Cloning and characterization of the glucosidase II αsubunit gene of Trichoderma reesei: a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30. Appl Environ Microbiol 2005, 71(6):2910-2924. 28. Seidl V, Gamauf C, Druzhinina IS, Seiboth B, Hartl L, Kubicek CP: The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome. BMC Genomics 2008, 9:327. 29. Nakari-Setälä T, Paloheimo M, Kallio J, Vehmaanperä J, Penttilä M, Saloheimo M: Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production. Appl Environ Microbiol 2009, 75(14):4853-4860. 30. Penttilä M, Limón C, Nevalainen H: Molecular Biology of Trichoderma and Biotechnological Applications. In Handbook of Fungal Biotechonology. Volume 20. Edited by: Arora DK. New York, Basel Marcel Dekker Inc; 2004:413-427. 31. Margolles-Clark E, Harman GE, Penttilä M: Enhanced expression of endochitinase in Trichoderma harzianum with the cbh1 promoter of Trichoderma reesei.Appl Environ Microbiol 1996, 62(6):2152-2155. 32. Valkonen M, Ward M, Wang H, Penttilä M, Saloheimo M: Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl Environ Microbiol 2003, 69(12):6979-6986. 33. Collén A, Saloheimo M, Bailey M, Penttilä M, Pakula TM: Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag. Biotechnol Bioeng 2005, 89(3):335-344. 34. Xu Q, Singh A, Himmel ME: Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose. Curr Opin Biotechnol 2009, 20(3):364-371. 35. Berg JM, Tymoczko JL, Stryer L: Biochemistry. New York: W.H. Freeman and Company, 5 2002. 36. Canonaco F, Hess TA, Heri S, Wang T, Szyperski T, Sauer U: Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact on overexpression of the soluble transhydrogenase UdhA. FEMS Microbiol Lett 2001, 204:247-252. 37. González-Siso MI, Freire Picos MA, Cerdán ME: Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants. FEBS Lett 1996, 387(1):7-10. 38. Aguilera A: Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae.Mol Gen Genet 1986, 204(2):310-316. 39. Maitra PK: Glucose and fructose metabolism in a phosphoglucoisomeraseless mutant of Saccharomyces cerevisiae. J Bacteriol 1971, 107:759-769. 40. Boles E, Lehnert W, Zimmermann FK: The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur J Biochem 1993, 217(1):469-477. 41. Ciriacy M, Breitenbach I: Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae.J Bacteriol 1979, 139:152-160. 42. Heux S, Cadiere A, Dequin S: Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains. FEMS Yeast Res 2008, 8(2):217-224. 43. Trichoderma reesei database v2.0. [http://genome.jgi-psf.org/Trire2/Trire2. home.html]. 44. Blast. [http://ncbi.nih.gov/BLAST/]. 45. Poulsen BR, Nøhr J, Douthwaite S, Hansen LV, Iversen JJL, Visser J, Ruijter GJG: Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger.FEBS Journal 2005, 272(6):1313-1325. 46. Marx A, Hans S, Mockel B, Bathe B, de Graaf AA, McCormack AC, Stapleton C, Burke K, O’Donohue M, Dunican LK: Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol 2003, 104(1-3):185-197. 47. Upadhyay S, Shaw BD: A phosphoglucose isomerase mutant in Aspergillus nidulansis defective in hyphal polarity and conidiation. Fungal Gen Biol 2006, 43:739-751. 48. Madrid MP, Di Pietro A, Roncero MI: Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds. Mol Microbiol 2003, 47(1):257-266. 49. Martín-Udiroz M, Madrid MP, Roncero MI: Role of chitin synthase genes in Fusarium oxysporum.Microbiology 2004, 150(Pt 10):3175-3187. 50. Fillinger S, Ruijter G, Tamas MJ, Visser J, Thevelein JM, d’Enfert C: Molecular and physiological characterization of the NAD-dependent glycerol 3- phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans.Mol Microbiol 2001, 39(1):145-157. 51. Fang W, Yu X, Wang B, Zhou H, Ouyang H, Ming J, Jin C: Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis. Microbiology 2009, 155(Pt 10):3281-3293. 52. Milewski S, Gabriel I, Olchowy J: Enzymes of UDP-GlcNAc biosynthesis in yeast. Yeast 2006, 23(1):1-14. 53. Pitkänen JP, Törmä A, Alff S, Huopaniemi L, Mattila P, Renkonen R: Excess mannose limits the growth of phosphomannose isomerase PMI40 deletion strain of Saccharomyces cerevisiae.J Biol Chem 2004, 279(53):55737-55743. 54. Katz ME, Bernardo SM, Cheetham BF: The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: evidence for a role for CreA in the response to carbon starvation. Curr Genet 2008, 54(1):47-55. 55. Morita T, El-Kazzaz W, Tanaka Y, Inada T, Aiba H: Accumulation of glucose 6-phosphate or fructose 6-phosphate is responsible for destabilization of glucose transporter mRNA in Escherichia coli.J Biol Chem 2003, 278(18):15608-15614. 56. Lee AT, Cerami A: Elevated glucose 6-phosphate levels are associated with plasmid mutations in vivo.Proc Natl Acad Sci USA 1987, 84(23):8311-8314. 57. Levi B, Werman MJ: Fructose triggers DNA modification and damage in an Escherichia coli plasmid. J Nutr Biochem 2001, 12(4):235-241. 58. Portnoy T, Margeot A, Seidl-Seiboth V, Le Crom S, Ben Chaabane F, Linke R, Seiboth B, Kubicek CP: Differential regulation of the cellulase transcription factors XYR1, ACE2 and ACE1 in high and low cellulase producing strains of Trichoderma reesei.Eukaryot Cell 2011, 10:262-271. 59. Fekete E, Karaffa L, Kubicek CP, Szentirmai A, Seiboth B: Induction of extracellular beta-galactosidase (Bga1) formation by D-galactose in Hypocrea jecorina is mediated by galactitol. Microbiology 2007, 153(Pt 2):507-512. 60. Aguilera A: Mutations suppressing the effects of a deletion of the phosphoglucose isomerase gene PGI1 in Saccaromyces cerevisiae.Curr Genet 1987, 11:429-434. 61. Gamo FJ, Portillo F, Gancedo C: Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae.FEMS Microbiol Lett 1993, 106(3):233-237. 62. Montenecourt BS, Eveleigh DE: Preparation of mutants of Trichoderma reesei with enhanced cellulase production. Appl Environ Microbiol 1977, 34(6):777-782. 63. Bailey MJ, Nevalainen KMH: Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb Technol 1981, 3:153-157. 64. Aro N, Saloheimo A, Ilmén M, Penttilä M: ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei.J Biol Chem 2001, 276(26):24309-24314. Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 20 of 21 65. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J: A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.Gene 1987, 61(2):155-164. 66. Sambrook J, Fritch EF, Maniatis M: Molecular cloning: A laboratory manual. Cold Spring, N.Y.: Cold Spring Laboratory;, 2 1989. 67. Rodríguez-Ortiz R, Limón MC, Avalos J: Regulation of carotenogenesis and secondary metabolism by nitrogen in wild-type Fusarium fujikuroi and carotenoid-overproducing mutants. Appl Environ Microbiol 2009, 75(2):405-413. 68. Pakula TM, Uusitalo J, Saloheimo M, Salonen K, Aarts RJ, Penttilä M: Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein. Microbiology 2000, 146(Pt 1):223-232. 69. Maitra PK, Lobo Z: A kinetic study of glycolytic enzyme synthesis in yeast. J Biol Chem 1971, 246:475-488. 70. van Tilbeurg H, Claeyssens M, de Bruyne C: The use of 4- methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes. FEBS Lett 1982, 149(1):152-156. 71. Limón MC, Pintor-Toro JA, Benítez T: Increased antifungal activity of Trichoderma harzianum transformants that overexpress a 33-kDa chitinase. Phytopathology 1999, 89(3):254-261. doi:10.1186/1475-2859-10-40 Cite this article as: Limón et al.: The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30. Microbial Cell Factories 2011 10:40. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Limón et al.Microbial Cell Factories 2011, 10:40 http://www.microbialcellfactories.com/content/10/1/40 Page 21 of 21