High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

Taina Karhunen, Arja Mäntylä, K. M Helena Nevalainen, Pirkko L. Suominen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

60 Citations (Scopus)

Abstract

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.

Original languageEnglish
Pages (from-to)515-522
Number of pages8
JournalMGG Molecular & General Genetics
Volume241
Issue number5-6
DOIs
Publication statusPublished - Dec 1993
Externally publishedYes

Keywords

  • Cellulases
  • Gene replacement
  • Homologous recombination
  • Regulation
  • Trichoderma reesei

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