The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose-inducible beta-glucosidase involved in cellulase induction by sophorose.

We have investigated the effect of disruption of the bgl1-(beta-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other beta-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75 kDa extracellular beta-glucosidase on cellulose or lactose, but still formed beta-glucosidase activity on glucose, cellobiose, xylan or beta-1,3-glucan, suggesting that the enzyme(s) exhibiting this beta-glucosidase activity is (are) not encoded by bgl1. The cellulase-inducer sophorose induced the bgl1-encoded beta-glucosidase, whereas the remaining beta-glucosidase activity was induced by methyl-beta-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other beta-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase l formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The beta-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded beta-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-beta-glucoside inducible beta-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.

Crystallization and preliminary X-ray analysis of two major xylanases from Trichoderma reesei.

Two major endoxylanases, endo-beta-1,4-xylanase I and II (molecular mass 19 kDa and 21 kDa) from the filamentous fungus Trichoderma reesei have been crystallized by using ammonium sulphate as the precipitating agent. Both crystals were monoclinic and belonged to the space groups C2 (a = 71.9 A, b = 39.0 A, c = 59.9 A, beta = 118.0 degrees, for XYNI) and P2(1) (a = 81.6 A, b = 60.6 A, c = 38.3 A, beta = 94.4, for XYNII). The crystals diffract to at least 2.2 A and 1.5 A, respectively.

Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei.

We have engineered the filamentous fungus Trichoderma reesei to assemble and secrete immunologically authentic engineered Fab antibody fragments into the culture medium. A major improvement in yield was achieved by fusing the heavy Fd chain to the T. reesei cellulase, CBHI. The yields of secreted, immunologically active Fab and CBHI-Fab fusion were 1 mg/l and 150 mg/l, respectively. The Fab fragment can be released from the fusion protein CBHI-Fab by an extracellular T. reesei protease. There was no detectable difference in affinity for the antigen between the engineered Fab and the idiotypic antibody.

The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes.

As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, we have purified two endo-beta-1,4-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and cloned their genes. The enzymes were isolated from culture filtrates of T. reesei C30 grown on xylan as a carbon source, using two steps of cation exchange chromatography. They exhibited molecular weights of 19 (XYN I) and 21 (XYN II) kD, and isoelectric points of 5.2 and 9.0, respectively. These enzymes differed in their pH optimum for activity and affinity for xylan, and accounted for more than 90% of the total xylanolytic activity of the fungus. The purified enzymes were subjected to N-terminal sequence analysis, and after cleavage with trypsin and endoproteinase Glu-C the resulting peptides were sequenced. Oligonucleotides based on these sequences were used to clone gene fragments via PCR, and these were used as probes to isolate full-length copies of xyn1 and xyn2 from a lambda gene bank of T. reesei. The products of xyn1 and xyn2 share considerable homology, but the enzyme encoded by xyn2 appears to more closely resemble several other bacterial and fungal xylanases than does that of xyn1.

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.

Use of lambda vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2.

A novel plasmid vector, pAMH70 carrying both the lamB and nusA genes of Escherichia coli K12 was constructed. Introduction of this plasmid into Salmonella typhimurium LT2 renders this bacterium both sensitive to lambda adsorption and able to sustain growth and lysogenization by lambda. Using this strain as a recipient, stable gene fusions to the gene encoding a major outer membrane porin protein OmpC, were constructed with a lambda vehicle lambda placMu. To confirm the actual site of fusions they were genetically mapped and transducing phages carrying the ompC-lacZ fusion were isolated and relysogenized. The fusions were also shown to be to ompC by their regulatory properties.

Expression of the Escherichia coli lamB gene in Vibrio cholerae.

A phage lambda mediated transduction system was devised to facilitate molecular analysis of Vibrio cholerae. A lamB expression plasmid, pAMH62 was introduced into Vibrio cholerae by conjugation. The resulting V. cholerae derivatives harboring pAMH62 produced substantial amounts of the LamB protein. This protein was properly inserted into the outer membrane, as suggested by (i) its localization into the cell envelope, (ii) its association with the peptidoglycan layer of the cell wall, and (iii) its function as receptor for phage lambda. In vivo packaged cosmids were efficiently transduced into these strains of V. cholerae.

Application of phage lambda technology to Salmonella typhimurium. Construction of a lambda-sensitive Salmonella strain.

We have previously constructed a novel strain of S. typhimurium carrying the E. coli lamB gene and shown that this strain adsorbs phage lambda and, for example, can be used for transposon mutagenesis with lambda vectors. In this study, we show that this strain can support the lytic growth of phage lambda nin derivatives, but not growth of wild-type lambda. However, lysogenization with lambda nin does not occur. Using this strain as starting material we took the construction one step further by introducing the E. coli nusA gene in a multicopy plasmid to this strain. We could show that this new Salmonella derivative can support both the lytic and lysogenic mode of growth of several different lambda derivatives. Using the same approach it should be possible to construct lambda-sensitive derivatives of other enteric bacteria thus rendering them more amenable to in vivo genetic manipulation.