High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction.
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.
Karhunen
T
T
Research Laboratories, Alko Ltd, Helsinki, Finland.
Mäntylä
A
A
Nevalainen
K M
KM
Suominen
P L
PL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D004274:DNA, Recombinant; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014170:Transformation, Genetic; D014242:Trichoderma / Q000235:genetics*
D004274:DNA, Recombinant; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D002480:Cellulase