Expression of Trichoderma koningii G-39 cellobiohydrolase I gene in Saccharomyces cerevisiae
| Autor: | Pi-Chu Chang, 張碧珠 |
|---|---|
| Rok vydání: | 1999 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 87 Cellobiohydrolase I (CBH I) is one of cellulose hydrolysis enzymes (cellulases). CBH I catalyzes the exocellulolytic cleavage of β-1,4-glucosidic bonds in cellulose and release cellobiose. For a long time, Cellulases have been used in commercial processes for the partial hydrolysis and softening of crude cellulose in food and cotton. Saccharomyces cerevisiae is an ideal organism for production of commercially important products from cellulose, a relatively cheap carbon source. However, S. cerevisiae can't directly utilize cellulose as carbon source. The aim of this study is to allow a stable expression of CBH I gene in the S. cerevisiae. Recombinant plasmid named pRUC1 was constructed in this investigation. pRUC1 contains CBH I cDNA gene from Trichoderma koningii G-39, portion of S. cerevisiae ribosomal DNA (rDNA), S. cerevisiae ura3 gene with a partially deleted promoter, and bacterial cloning vector pSP72. The rDNA of recombinant plasmid was used to target the homologous recombination in yeast chromosome, while marker gene with a partially deleted promoter renders high copy number of integration of transformed plasmid under selection pressure. pRUC1 was transformed into S. cerevisiae. A part of transformants were able to form clear zone on the plate containing 0.5% soluble carboxymethylcellulose (CMC), indicating the secretion of CBH I by the yeast. Southern hybridization analysis revealed that plasmids were integrated into S. cerevisiae chromosome as tandem repeats and dispersed copies. The Ura+ phenotype of transformants was stably maintained in the nonselective medium for 50 generations. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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