Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Development of a transformation system forTrichoderma longibrachiatum and its use for constructing multicopy transformants for theegl1 gene

  • 56 Accesses


An efficient transformation system for the fungusTrichoderma longibrachiatum has been developed. Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying theEscherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker. The transformation frequency was 0.5 to 5 transformants /μg plasmid DNA. Transformation normally occurred by tandem integration of the transforming DNA. A high percentage of the transformants were mitotically unstable. The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of theegll gene encoding a β-(1,4)-endoglucanase were obtained. Some of them secrete increased levels of endoglucanase to the culture medium. In addition, theE. coli lacZ gene was expressed in an active form under control of theAspergillus nidulans gpdA gene promoter.

This is a preview of subscription content, log in to check access.


  1. Arsdell JN van, Kwok S, Schweickart VL, Ladner M, Gelfland DH, Innis MA (1987) Cloning, characterization, and expression inSaccharomyces cerevisiae of endoglucanase I fromTrichoderma reesei. Bio/Technology 5: 60–64

  2. Barnett CC, Berka RM, Fowler T (1991) Cloning and amplification of the gene encoding an extracellular β-glucosidase fromTrichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Bio/Technology 9: 562–567

  3. Bergès T, Barreau C (1991) Isolation of uridine auxotrophs fromTrichoderma reesei and efficient transformation with the clonedura3 andura5 genes. Curr Genet 19: 359–365

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254

  5. Chen MC, Gritzali M, Stafford WD (1987) Nucleotide sequence and deduced primary structure of cellobiohydrolase II fromTrichoderma reesei. Bio/Technology 5: 274–278

  6. Cheng C, Tsukagoshi N, Udaka S (1990) Transformation ofTrichoderma viride using theNeurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene fromAspergillus oryzae. Curr Genet 18: 453–456

  7. Durand H, Baron M, Calmels T, Tiraby G (1988) Classical and molecular genetics applied toTrichoderma reesei for the selection of improved cellulolytic industrial strains. In: Paubert JP, Beguin P, Millet J (eds) Biochemistry and genetics of cellulose degradation. Academic Press, London, pp 135–151

  8. Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction fragments to high specific activity. Anal Biochem 132: 6–13

  9. Goldman GH, Montagu M van, Herrera-Estrella A (1990) Transformation ofTrichoderma harzianum by high-voltage electric pulse. Curr Genet 17: 169–174

  10. González R, Ferrer S, Buesa J, Ramón D (1989) Transformation of the dermatophyteTrichophyton mentagrophytes to hygromycin B resistance. Infect Immun 57: 2923–2925

  11. González R, Ramón D, Pérez-González JA (1992) Cloning, sequence analysis and yeast expression of theegll gene fromTrichoderma longibrachiatum. Appl Microbiol Biotechnol 38: 370–375

  12. Gorcom RFM, Pouwels PH, Goosen T, Visser J, Broek HWJ van der, Hamer JE, Timberlake WE, Hondel CAMJJ van den (1985) Expression of anEscherichia coli β-galactosidase fusion gene inAspergillus nidulans. Gene 40: 99–106

  13. Gruber F, Visser J, Kubicek CP, Graaf L de (1990a) The development of a heterologous transformation system for the cellulolytic fungusTrichoderma reesei based on apyrG-negative mutant strain. Curr Genet 18: 71–76

  14. Gruber F, Visser J, Kubicek CP, Graaf L de (1990b) Cloning of theTrichoderma reesei pyrG gene and its use as a homologous marker for a high-frequency transformation system. Curr Genet 18: 447–451

  15. Hanahan D (1983) Studies on transformation ofEscherichia coli with plasmids. J Mol Biol 166: 557–580

  16. Herrera-Estrella A, Goldman GH, Montagu M van (1990) High-efficiency transformation system for the biocontrol agents,Trichoderma spp. Mol Microbiol 4: 839–843

  17. Knowles JKC, Lehtovaara P, Teeri T, Penttillä ME, Salovouri T, André L (1987) The application of recombinant DNA technology to cellulases and lignocellulosic wastes. Philos Trans R Soc London [Biol] 321: 449–454

  18. Mandels M (1985) Application of cellulases. Biochem Soc Trans 13: 414–416

  19. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, New York

  20. Montenecourt BS (1983)Trichoderma reesei cellulases. Trends Biotechnol 1: 156–161

  21. Myers AM, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction oflacZ fusions. Gene 45: 299–310

  22. Papavizas GC (1985)Trichoderma andGliocladium: biology, ecology, and potential for biocontrol. Annu Rev Phytopathol 23: 23–54

  23. Penttillä ME, Lehtovaara P, Nevalainen H, Bhikhabhai R, Knowles JKC (1986) Homology between cellulase genes ofTrichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45: 253–263

  24. Penttillä ME, André L, Saloheimo M, Lehtovaara P, Knowles JKC (1987a) Expression of twoTrichoderma reesei endoglucanases in the yeastSaccharomyces cerevisiae. Yeast 3: 175–185

  25. Penttillä ME, Nevalainen H, Rattö M, Salminen E, Knowles JKC (1987b) A versatile transformation system for the cellulolytic filamentous fungusTrichoderma reesei. Gene 61: 155–164

  26. Pérez-González JA, González R, Querol A, Sendra J, Ramón D (1993) Construction of a recombinant wine yeast strain expressing a β-(1,4)-endoglucanase activity and its use in microvinification experiments. Appl Environ Microbiol 59: 2801–2806

  27. Pontecorvo G, Ropper JA, Jemmons LJ, MacDonald KD, Buft AWJ (1953) The genetics ofAspergillus nidulans. Adv Genet 5: 141–238

  28. Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Hondel CAMJJ van den (1987) Transformation ofAspergillus based on the hygromycin B resistance marker fromEscherichia coli. Gene 56: 117–124

  29. Ruiz-Sala P, Pérez-González JA, Ramón D (1993) Nucleotide sequence of aTrichoderma longibrachiatum DNA fragment encoding the 5.8S rRNA gene. Nucleic Acids Res 21: 741

  30. Saloheimo M, Lehtovaara P, Penttillä ME, Teeri TT, Stahlberg J, Petterson G, Claeyssens M, Tomme P, Knowles JK (1988) EGIII, a new endoglucanase fromTrichoderma reesei: the characterization of both gene and enzyme. Gene 63: 11–21

  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

  32. Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kowk S, Myambo K, Innis M (1983) Molecular cloning of exo-cellobiohydrolase I derived fromTrichoderma reesei strain L27. Bio/technology 1: 691–696

  33. Sivan A, Stasz TE, Hemmat M, Hayes CK, Harman GE (1992) Transformation ofTrichoderma spp. with plasmids conferring hygromycin B resistance. Mycologia 84: 687–694

  34. Smith JL, Bayliss FT, Ward M (1991) Sequence of the clonedpyr4 gene ofTrichoderma reesei and its use as homologous selectable marker for transformation. Curr Genet 19: 27–33

  35. Sternberg D (1976) Production of cellulase byTrichoderma. Biotechnol Bioeng Symp 6: 35–53

  36. Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes inTrichoderma reesei by sophorose. J Bacteriol 139: 761–769

  37. Teeri TT, Lehtovaara P, Kauppinen S, Salovouri I, Knowles JKC (1987) Homologous domains inTrichoderma reesei cellulolytic enzymes:gene sequence and expression of cellobiohydrolase II. Gene 51: 43–52

  38. Ulhoa CJ, Vainstein MH, Peberdy JF (1992) Transformation ofTrichoderma species with dominant selectable markers. Curr Genet 21: 23–26

  39. Ventura L, Ramón D (1991) Transformation ofAspergillus terreus with the hygromycin B resistance marker fromEscherichia coli. FEMS Microbiol Lett 82: 189–194

  40. Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Methods Enzymol 160: 87–112

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sánchez-Torres, P., González, R., Pérez-González, J.A. et al. Development of a transformation system forTrichoderma longibrachiatum and its use for constructing multicopy transformants for theegl1 gene. Appl Microbiol Biotechnol 41, 440–446 (1994). https://doi.org/10.1007/BF00939033

Download citation


  • Active Form
  • Gene Promoter
  • Hygromycin
  • Efficient Transformation
  • Multiple Copy