HIGH-FREQUENCY ONE-STEP GENE REPLACEMENT IN TRICHODERMA-REESEI .1. ENDOGLUCANASE-I OVERPRODUCTION

The chromosomal cellobiohydrolase I locus (cbh1) of the biotechnologic ally important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglu canase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting o f the linear expression cassette to the cbh1 locus was efficient. Stud ies of expression of the intact cbh1-egl1 cassette at the chb1 locus r evealed that egl1 cDNA is expressed from the cbh1 promoter as efficien tly as cbh1 itself. Furthermore, a strain carrying two copies of the c bh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. Th e level of egl1-specific mRNA in the single-copy transformant was abou t 10-fold higher than that found in the non transformed host strain, i ndicating that the cbh1 promoter is about 10 times stronger than the e gl1 promoter. The 10-fold increase in the secreted EG I protein, measu red with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.