High frequency one-step gene replacement in Trichoderma reesei. 1. Endoglucanase 1 overproduction
1993
Karhunen, T. (Alko Ltd., Helsinki (Finland). Research Labs.) | Maentylae, A. | Nevalainen, K.M.H. | Suominen, P.L.
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 1 cDNA (egl1) under control of the cbh1 promoter. CBH1 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 EG1 as the amount of CBH1, 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 EG1 protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.
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