Development of stable flocculent Saccharomyces cerevisiae strain for continuous...

• Author(s): Oliveira, Carla Cristina Marques de ; Teixeira, J. A. ; Lima, Nelson ; Silva, Nancy A. da ; Domingues, Lucília
• Date: 2007
• Persistent ID: http://hdl.handle.net/1822/8881
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Genetic stability of delta-integrating systems; Continuous high-cell-density culture; Aspergillus niger β-galactosidase production; Recombinant Saccharomyces cerevisiae; Yeast flocculation
• Document Type: Article
• Language: English

Description

A flocculent Saccharomyces cerevisiae strain was engineered to stably secrete Aspergillus niger β-galactosidase in a continuous high-cell-density bioreactor. The δ-sequences from the yeast retrotransposon Ty1 were used as target sites for the integration of the β-galactosidase expression cassette. High-copy-number transformants were successfully obtained using the δ-integration system together with the dominant selection antibiotic, G418. The integration of multiple copies was confirmed by genomic Southern blot analysis. Integrants with the highest β-galactosidase levels (approximately eight gene copies) had similar β-galactosidase activities as a recombinant strain carrying the β-galactosidase expression cassette in a YEp-based vector. The β-galactosidase expression cassettes integrated into the yeast genome were stably maintained after eight sequential batch cultures in a nonselective medium. In continuous high-cell-density culture under the same operating conditions, the integrant strain was more stable than the plasmid-carrying strain. To our knowledge, this is the first study of multicopy δ-integrant stability in a continuous bioreactor operating at different dilution rates.