The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.     

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

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 β-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.     

Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.     

Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens

Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation.     

Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.The filamentous fungus Trichoderma reesei (Hypocrea jecorina) produces large amounts of extracellular enzymes. The majority of the secreted proteins are various plant polymer-degrading enzymes; the most abundant of these enzymes are the cellobiohydrolases and endoglucanases that act synergistically to break down cellulose. This fungus has been used as a production host for various industrial enzymes, including products tailored for textile, feed, food, and pulp and paper applications (6, 10). It has been reported that protein production levels in the industrial T. reesei process exceed 100 g/liter (7).The major cellulase and hemicellulase genes are regulated in a coordinate manner by the carbon source available (2, 9, 14). Cellulose and other plant materials and other substances (for example, lactose) induce the expression of cellulase and hemicellulase genes, while glucose acts as a repressing carbon source. Several genes coding for regulators of cellulase and hemicellulase expression have been isolated. These include CREI mediating carbon catabolite repression, the repressor ACEI, the activator ACEII, the CCAAT binding complex Hap2/3/5 (reviewed in references 2, 17, and 27) and the activator XYRI (29). The CREI protein has sequence similarity with other fungal proteins mediating glucose repression, such as Aspergillus nidulans CREA (8) and Saccharomyces cerevisiae MIG1 and RGR1 (22). In T. reesei, glucose repression has been shown to occur upon binding of CREI to specific sequences in the cbh1 promoter (13). A mutant cre1 gene (cre1-1) encoding a truncated form of CREI has been isolated from the hypercellulolytic T. reesei strain Rut-C30, which is capable of cellulase and hemicellulase production on glucose-containing media. Further evidence for the function of CREI in glucose repression was obtained by complementation of the cre1-1 mutation of Rut-C30 by the wild-type cre1 gene, which restored the glucose-repressed phenotype of the strain (15).In this paper, we wanted to address three questions. (i) What is the effect of cre1 mutations in the wild-type background? (ii) Is cre1-1 a null mutation? (iii) Can enzyme production be further improved by cre1 deletion in an industrial production strain improved greatly by mutagenesis and screening programs? Therefore, we introduced cre1-1 allele and cre1 deletion to the wild-type strain QM6a and the cre1 deletion into the industrial strain VTT-D-80133 and studied the effects of these mutations on enzyme production.     

Intraspecific hybridization of Trichoderma reesei by protoplast fusion

Protoplasts obtained from mycelia of a single auxotrophic mutant of Trichoderma reesei QM 9414 were fused with those of T. reesei QM 9136 in the presence of 0.5 M glycine-NaOH buffer, pH 7.5, containing 0.05 M CaCl₂ · 2H₂O and 35% polyethylene glycol 4,000. The regeneration frequency of these protoplasts was 8.9–12.0% on a solid culture medium with soft agar overlay. The fused protoplasts successfully formed heterokaryons showing 3.33% of the fusion frequency. A heterozygous diploid was obtained from conidia of the heterokaryon by treatment with 0.1% d-camphor. The diploid showed a 1.9 fold DNA content per conidial nucleus compared to T. reesei QM 9414. The frequency of diploid formation was about 1.9 × 10⁻⁴ per conidium. Cellulase activities, such as filter paper degrading and CM-cellulose and Avicel saccharifying activities, and the xylanase activity of the diploid showed intermediate values between those of T. reesei QM 9414 and T. reesei QM 9136. However, the β-glucosidase, β-1,3-glucanase and chitinase activities of the diploid increased to levels equal to on above those of T. reesei QM 9414 and T. reesei QM 9136. The existence of a parasexual cycle of T. reesei and the possibility of its application to enhanced enzyme productivity were confirmed using the protoplast fusion technique.     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.     

Spent brewery grains as substrate for the production of cellulases byTrichoderma reesei QM9414

Summary The cellulolytic fungusTrichoderma reesei QM9414 can be cultivated on spent brewery grains for the production of cellulases. The levels of the cellulase components endoglucanase and exoglucanase synthesized, and the complexes filter paper cellulase and grain-hydrolyzing cellulase synthesized by the organism on spent grains were as high as 287, 182, 187, and 449 units per g available cellulose, respectively. Scaling up the spent grains fermentation system by up to 40-fold (200g dry substrate/tray) demonstrated that cellulase production was comparable to laboratory scale (5g dry substrate/flask) yields. Cultivation of the fungus was feasible on spent grains without pretreatment or further adjustment, although the enzyme yield was somewhat lower than that on dried grains moistened with water orTrichoderma medium. This suggested the possible reutilization of spent grains, with minimal pretreatment, in the cultivation ofT.reesei QM9414 for cellulase synthesis and for future incorporation into animal feed.     

Purification and characteristics of xyloglucanase and five other cellulolytic enzymes from Trichoderma reesei QM9414

By combining anion-exchange chromatography with gel filtration, an effective method for purification of wild-type xyloglucanase and five other cellulolytic enzymes from strain QM9414 of Trichoderma reesei was established. Characterization by enzyme activity assay, SDS-PAGE, and mass spectrometry identified the purified proteins as cellobiohydrolases I and II, endoglucanases I and II, a xyloglucanase, and β-xylosidase, of which the xyloglucanase was purified for the first time from the mutant strain QM9414. This method holds great promise to study the mechanism of cellulolytic enzymes, to investigate the synergistic action between cellulase and other cellulolytic enzymes, and to better exploit enzyme preparations for degradation of lignocellulose.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cerevisiae

The combined effect of pH and temperature on carboxymethyl cellulase from two intergeneric fusants (M 14 and M 62) of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 was studied using response surface methodology. A central composite design for two variables was employed for the optimization studies. This study was compared with similar studies carried out with Trichoderma reesei QM 9414. The optimal pH and temperature for the enzymes derived from these organisms were: for the fusant M 14—pH 5.7 and 41.7°C, for the fusant M 62—pH 5.3 and 43°C, and for Trichoderma reesei QM 9414—pH 4.31 and 38.3°C. Received 5 May 1997/ Accepted in revised form 17 July 1998     

Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei

Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18 % compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml^?1, while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml^?1, respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml^?1, respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.