Abstract
Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aho S, Olkkonen V, Jalava T, Paloheimo M, Bühler R, Niku-Paavola M-L, Bamford DH, Korhola M (1991) Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. Eur J Biochem 200:643–649
Bailey MJ, Nevalainen KMH (1981) Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb Technol 3:153–157
Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270
Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagro LP, Thompson SA (1990) Molecular cloning and deletion of the gene coding aspergillopepsin. Gene 86:153–162
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bullock WO, Fernandez JM, Short JM (1987) XL1-Blue: A high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5:376–378
Bühler R (1991) Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei. Appl Environ Microbiol 57:3317–3321
Esser K, Mohr G (1986) Integrative transformation of filamentous fungi with respect to biotechnological application. Process Biochem 20:153–159
Finkelstein DB (1992) Transformation. In: Finkelstein DB, Ball C (eds) Biotechnology of filamentous fungi. Technology and products. Butterworth-Heinemann, Boston, pp 113–156
Harkki A, Mäntylä A, Penttilä M, Muttilainen S, Bühler R, Suominen P, Knowles J, Nevalainen H (1991) Genetic engineering of Trichoderma to produce strains with novel cellulase profiles. Enzyme Microb Technol 13:227–233
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Karhunen T, Mäntylä A, Nevalainen KMH, Suominen PL (1993) High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet 241:515–522
Kelly JM, Hynes MJ (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479
Lowry OH, Roseborough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Mandels M, Andreotti R, Roche C (1976) Measurement of saccharifying cellulase. In: Gaden EL, Mandels MH, Reese ET, Spano LA (eds) Biotechnology Bioengineering Symposium 6. Wiley, New York, pp 21–33
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Nevalainen KMH, Palva ET (1978) Production of extracellular enzymes in mutants isolated from Trichoderma viride unable to hydrolyze cellulase. Appl Environ Microbiol 35:11–16
Penttilä M, Lehtovaara P, Nevalainen H, Bhikhabhai R, Knowles J (1986) Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45:253–263
Penttilä M, André L, Saloheimo M, Lehtovaara P, Knowles JKC (1987a) Expression of two Trichoderma reesei endoglucanases in yeast Saccharomyces cerevisiae. Yeast 3:175–185
Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987b) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164
Penttilä ME, André L, Lehtovaara P, Bailey M, Teeri TT, Knowles JKC (1988) Efficient secretion of two fungal cellobiohydrolases in Saccharomyces cerevisiae. Gene 63:103–112
Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20
Saloheimo M, Lehtovaara P, Penttilä M, Teeri TT, Stålberg J, Johansson G, Pettersson G, Claeyssens M, Tomme P, Knowles JKC (1988) EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63:11–21
Seiboth B, Messner R, Gruber F, Kubicek CP (1992) Disruption of the Trichoderma reesei cbh2 gene coding for cellobiohydrolase II leads to a delay in the triggering of cellulase formation by cellulose. J Gen Microbiol 138:1259–1264
Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis M (1983) Molecular cloning of exo-cellobiohydrolase from Trichoderma reesei strain L27. Bio/Technology 1:691–696
Suominen P, Mäntylä A, Saarelainen R, Paloheimo M, Fagerström R, Parkkinen E, Nevalainen H (1992) Genetic engineering of Trichoderma reesei to produce suitable enzyme combinations for applications in the pulp and paper industry. In: Kuwahara M, Shimada M (eds) Biotechnology in pulp and paper industry. Uni Publishers, Tokyo, Japan, pp 439–445
Teeri T, Salovuori I, Knowles J (1983) The molecular cloning of the major cellulase gene from Trichoderma reesei. Bio/Technology 1:696–699
Teeri T, Lehtovaara P, Kauppinen S, Salovuori I, Knowles J (1987) Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51:43–52
van Arsdell JN, Kwok S, Schweickart VL, Ladner MB, Gelfand DH, Innis MA (1987) Cloning, characterization and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Bio/Technology 5:60–64
Woodward (1989) Immobilized cellulases for cellulose utilization. J Biotechnol 11:299–312
Author information
Authors and Affiliations
Additional information
Communicated by C. van den Hondel
Rights and permissions
About this article
Cite this article
Suominen, P.L., Mäntylä, A.L., Karhunen, T. et al. High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. Molec. Gen. Genet. 241, 523–530 (1993). https://doi.org/10.1007/BF00279894
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00279894