High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction
| Autor: | Arja Mäntylä, T Karhunen, Pirkko Suominen, K M Nevalainen |
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| Rok vydání: | 1993 |
| Předmět: |
Genes
Fungal Molecular Sequence Data DNA Recombinant Fungal Proteins Transformation Genetic Cellulase Complementary DNA Gene Expression Regulation Fungal Gene expression Genetics Amino Acid Sequence RNA Messenger Overproduction Promoter Regions Genetic Molecular Biology Gene Trichoderma reesei Regulation of gene expression Trichoderma biology Base Sequence Fungal genetics RNA Fungal biology.organism_classification Molecular biology Expression cassette Genetic Engineering |
| Zdroj: | Moleculargeneral genetics : MGG. 241(5-6) |
| ISSN: | 0026-8925 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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