Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain

Appl Microbiol Biotechnol. 2004 Jun;64(6):833-9. doi: 10.1007/s00253-003-1547-5. Epub 2004 Jan 23.

Abstract

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Cellulase / chemistry
  • Cellulase / metabolism
  • Cellulose / chemistry
  • Cellulose / metabolism
  • Geobacillus stearothermophilus / enzymology*
  • Lipase / biosynthesis*
  • Lipase / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional / methods
  • Proprotein Convertases / genetics
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Time Factors
  • Trichoderma

Substances

  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Cellulose
  • Lipase
  • endoglucanase 2
  • Cellulase
  • Proprotein Convertases
  • KEX2 protein, S cerevisiae