title: Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger beta-galactosidase production.
abstract: A flocculent Saccharomyces cerevisiae strain was engineered to stably secrete Aspergillus niger beta-galactosidase in a continuous high-cell-density bioreactor. The delta-sequences from the yeast retrotransposon Ty1 were used as target sites for the integration of the beta-galactosidase expression cassette. High-copy-number transformants were successfully obtained using the delta-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 beta-galactosidase levels (approximately eight gene copies) had similar beta-galactosidase activities as a recombinant strain carrying the beta-galactosidase expression cassette in a YEp-based vector. The beta-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 delta-integrant stability in a continuous bioreactor operating at different dilution rates.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Carla
authors.initials: C
authors.lastname: Oliveira
authors.forename: José A
authors.initials: JA
authors.lastname: Teixeira
authors.forename: Nelson
authors.initials: N
authors.lastname: Lima
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Da Silva
authors.forename: Lucília
authors.initials: L
authors.lastname: Domingues
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D019149:Bioreactors / Q000382:microbiology; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D007218:Industrial Microbiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase