Applied Biochemistry and Biotechnology

, Volume 87, Issue 1, pp 1–15 | Cite as

Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica

  • Cheon Seok Park
  • Ching Chuan Chang
  • Dewey D. Y. Ryu


The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.

Index Entries

Gene expression secretion cellulase endoglucanase I high-cell density culture fed-batch fermentation Yarrowia lipolytica 

The abbreviations used in this article are




hydroxyethylcellulose linked Ostazin Brilliant Red H-3B


alkaline extracellular protease


endoplasmic reticulum


dinitrosalicylic acid


polymerase chain reaction


sodium dodecyl sulfate polyacrylamide gel electrophoresis

Endo H

endoglycosidase H


KEX2-like endopeptidase


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  1. 1.
    Buckholz, R. G. and Gleeson, M. A. G. (1991), Bio/Technology 9, 1067–1072.CrossRefGoogle Scholar
  2. 2.
    Buckholz, R. G. (1993), Curr. Opin. Biotechnol. 4, 538–542.CrossRefGoogle Scholar
  3. 3.
    Gellissen, G., Melber, K., Janowicz, Z. A., Dahlems, U. M., Weydemann, U., Piontek, M., Strasser, A. W., and Hollenberg, C. P. (1992), Antoine Van Leeuwenhoek 62, 79–93.CrossRefGoogle Scholar
  4. 4.
    Russell, C., Mawson, J., and Yu, P. L. (1991), Aust. J. Biotechnol. 5, 48–55.Google Scholar
  5. 5.
    Sudbery, P. E. (1996), Curr. Opin. Biotechnol. 7, 517–524.CrossRefGoogle Scholar
  6. 6.
    Chang, C. C., Ryu, D. D. Y., Park, C. S., Kim, J. Y., and Ogrydziak, D. M. (1998), Appl. Microbiol. Biotechnol. 49, 531–537.CrossRefGoogle Scholar
  7. 7.
    DeZeeuw, J. R. and Tynan, E. J. (1973a), USA Patent No 3736229.Google Scholar
  8. 8.
    DeZeeuw, J. R. and Tynan, E. J. (1973b), USA Patent No 3756917.Google Scholar
  9. 9.
    Heslot, H. (1990), Adv. Biochem. Eng. Biotech. 43, 43–73.Google Scholar
  10. 10.
    Rane, K. D. and Sims, K. A. (1994), Biotechnol. Bioeng. 43, 131–137.CrossRefGoogle Scholar
  11. 11.
    Tobe, S., Takami, T., Ikeda, S., and Mitsugi, K. (1976), Agric. Biol. Chem. 40, 1087–1092.Google Scholar
  12. 12.
    Matoba, S., Fukayama, J., Wing, R. A., and Ogrydziak, D. M. (1988), Mol. Cell. Biol. 8, 4904–4916.Google Scholar
  13. 13.
    Harkki, A., Mantyla, A., Penttila, M., Muttilainen, S., Buhler, R., Suominen, P., Knowles, J., and Nevalainen, H. (1991), Enzyme Microb. Technol. 13, 227–233.CrossRefGoogle Scholar
  14. 14.
    Niku-Paavola, M.-L., Lappalainen, A., Enari, T.-M., and Nummi, M. (1985), Biochem. J. 231, 75–81.Google Scholar
  15. 15.
    Beguin, P. and Aubert, J.-P. (1994), FEMS Microbiol. Rev. 13, 25–58.CrossRefGoogle Scholar
  16. 16.
    Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991), Microbiol. Rev. 55, 303–315.Google Scholar
  17. 17.
    Knowles, J., Pentilla, M., Teeri, T., Andre, L., Lehtovaara, P., and Salovuori, I. (1987), in Biological Research on Industrial Yeasts, Steward, G. G. et al., ed., CRC Press, Boca Raton, pp. 189–199.Google Scholar
  18. 18.
    Wong, W. K. R., Curry, C., Parekh, R. S., Parekh, S. R., Wayman, M., Davies, R. W., Kilburn, D. G., and Skipper, N. (1988), Bio/Technology 6, 713–719.CrossRefGoogle Scholar
  19. 19.
    Silva, A., Benitez, J., and Hollenberg, C. P. (1991), Anal. Biochem. 197, 290–295CrossRefGoogle Scholar
  20. 20.
    Hanahan, D. (1983), J. Mol. Biol. 166, 557–580.CrossRefGoogle Scholar
  21. 21.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory, New York.Google Scholar
  22. 22.
    Enderlin, C. S. and Ogrydziak, D. M. (1994), Yeast 10, 67–79.CrossRefGoogle Scholar
  23. 23.
    Farkas, V., Liskova, M., and Biely, P. (1985), FEMS Microbiol. Lett. 28, 137–140.CrossRefGoogle Scholar
  24. 24.
    Inoue, H., Nojima, H., and Okayama, H. (1990), Gene 96, 22–28.Google Scholar
  25. 25.
    Gaillardin, C., Ribet, A. M., and Heslot, H. (1985), Curr. Genet. 10, 49–58.CrossRefGoogle Scholar
  26. 26.
    Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989), Gene 77, 61–68.CrossRefGoogle Scholar
  27. 27.
    Laemmli, U. K. (1970), Nature (London) 227, 680–685.CrossRefGoogle Scholar
  28. 28.
    Bailey, M. J. and Nevalainen, K. M. H. (1981), Enzyme Microb. Technol. 3, 153–157.CrossRefGoogle Scholar
  29. 29.
    Owolabi, J. B., Beguin, P., Kilburn, D. G., Miller, R. C., and Warren, R. A. J. (1988), Appl. Environ. Microbiol. 54, 518–523.Google Scholar
  30. 30.
    Park, C. S., Chang, C. C., Kim, J. Y., Ogrydziak, D. M., and Ryu, D. D. Y. (1997), J. Biol. Chem. 272, 6876–6881.CrossRefGoogle Scholar
  31. 31.
    Franke, A. E., Kaczmarek, F. S., Eisenhard, M. E., Geoghegan, K. F., Danley, D. E., DeZeeuw, J. R., O’Donnell, M. M., Gollaher, M. G., Jr., and Davidow, L. S. (1988), Dev. Ind. Microbiol. 29, 43–57.Google Scholar
  32. 32.
    Tharaud, C., Ribet, A.-M., Costes, C., and Gaillardin, C. (1992), Gene 121, 111–119.CrossRefGoogle Scholar
  33. 33.
    Gaillardin, C., Fournier, C. P., Nicaud, J. M., He, F., Fabre, E., Lopez, M. C., and Tharaud, C. (1990), in Proceedings of the 6th International Symposium on Genetics of Industrial Microorganisms, Vol. 2, Heslot, H., Davies, J., Florent, J., Bobichon, L., Durand, G., and Penasse, L., eds., Société Française de Microbiologìe, Strasbourg, pp. 509–518.Google Scholar
  34. 34.
    Nicaud, J. M., Fournier, P., Bonnardiere, C. L., Chasles, M., and Gaillardin, C. (1991), J. Biotechnol. 19, 259–270.CrossRefGoogle Scholar
  35. 35.
    Penttila, M. E., Andre, L., Saloheimo, M., Lehtovaara, P., and Knowles, J. K. (1987), Yeast 3, 175–185.CrossRefGoogle Scholar
  36. 36.
    Van Arsdell, J. N., Kwok, S., Schweickart, V. L., Ladner, M. B., Gelfand, D. H., and Innis, M. A. (1987), Bio/Technology 5, 60–64.CrossRefGoogle Scholar
  37. 37.
    Lafontaine, D. and Tollervey, D. (1996), Nucleic Acids Res. 24, 3469–3472.CrossRefGoogle Scholar
  38. 38.
    Ledall, M. T., Nicaud, J. M., and Gaillardin, C. (1994), Curr. Genet. 26, 38–44.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Cheon Seok Park
    • 1
    • 2
  • Ching Chuan Chang
    • 1
  • Dewey D. Y. Ryu
    • 1
  1. 1.Department of Chemical Engineering and Material ScienceBiochemical Engineering ProgramUSA
  2. 2.Department of Food Science and TechnologyUniversity of CaliforniaDavis

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