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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
Article

Abstract

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

EGI

endoglucanase

OBR-HEC

hydroxyethylcellulose linked Ostazin Brilliant Red H-3B

AEP

alkaline extracellular protease

ER

endoplasmic reticulum

DNS

dinitrosalicylic acid

PCR

polymerase chain reaction

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Endo H

endoglycosidase H

Xpr6p

KEX2-like endopeptidase

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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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