Jari Vehmaanperä

Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production

ABSTRACT 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.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes. Biotechnol. Bioeng. 2008;101: 515–528.

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B.

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔTm = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔTm = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔTm = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single‐module, cellulose‐degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello‐oligomers: cellobiose (Glc2), cellotriose (Glc3), and cellotetraose (Glc4), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β‐sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello‐oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello‐oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C1) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.

Characterization of the bga1-encoded glycoside hydrolase family 35 β-galactosidase of Hypocrea jecorina with galacto-β-d-galactanase activity

The extracellular bga1‐encoded β‐galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI‐MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl‐ and alkyl‐β‐d‐galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o‐nitrophenyl‐β‐d‐galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is ∼ 5.0, and the optimum temperature was found to be 60 °C. Bga1 is also capable of releasing d‐galactose from β‐galactans and is thus actually a galacto‐β‐d‐galactanase. β‐Galactosidase is inhibited by its reaction product d‐galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto‐oligosaccharides.

Production of Industrial Enzymes in Trichoderma reesei

Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the two major fungal platforms for industrial enzyme production, along with Aspergillus sp. Its use derives from its history as a model organism for studies on cellulose degradation and its cellulase enzyme complex since the 1940s, which suggested its use for industrial bioethanol manufacturing during the oil crisis in the mid 1970s. Extensive strain development campaigns by different laboratories proved that the wild type isolate QM6a can be developed into superior production strains, and later the genetic tools were established for the species, which allowed the strains to be tailored for maximum productivity with optimised backgrounds. T. reesei has now maintained its position as a highly productive, easy-to-handle, robust and safe cell factory for more than 40 years. The recently revived interest in lignocellulosic bioethanol paved the way for a comeback in the use of T. reesei native cellulase complex, whereas advances in the development of molecular biology tools—such as bioinformatics and mating—have provided further refinements in the modern strain development of T. reesei for enzyme and other protein production in virtually all segments of industrial biotechnology.

Trichoderma Enzymes for Textile Industries

Abstract Introducing enzymes into the textile industry has been an environmentally sustainable approach, leading to high-quality products and cost savings in the processes. During the last three decades the use of enzymes has been fully accepted by the textile manufacturers, and there is still potential for novel and improved enzyme applications in future textile processing. Trichoderma cellulases have been the one of the pioneering enzyme products brought onto the market, and there was a concentration of extensive studies involving Trichoderma reesei enzymes at the turn of the millennium. Nowadays novel cellulase products from other fungal sources have proven useful in the textile industry, and T. reesei can be considered to be one of the most relevant production platforms for textile enzymes.

Preliminary X-ray analysis of cellobiohydrolase Cel7B from Melanocarpus albomyces

Cellobiohydrolases are enzymes that cleave off cellobiose units from cellulose chains in a processive manner. Melanocarpus albomyces Cel7B is a thermostable single-module cellobiohydrolase that has relatively low activity on small soluble substrates at room temperature. It belongs to glycoside hydrolase family 7, which includes endo-beta-1,4-glucanases and cellobiohydrolases. Cel7B was crystallized using the hanging-drop vapour-diffusion method and streak-seeding. The crystals belonged to space group P2(1), with unit-cell parameters a = 50.9, b = 94.5, c = 189.8 A, beta = 90.0 degrees and four monomers in the asymmetric unit. Analysis of the intensity statistics showed that the crystals were pseudo-merohedrally twinned, with a twinning fraction of 0.37. X-ray diffraction data were collected at 1.6 A resolution using synchrotron radiation.