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  • Title: Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger beta-galactosidase production.
    Author: Oliveira C, Teixeira JA, Lima N, Da Silva NA, Domingues L.
    Journal: J Biosci Bioeng; 2007 Apr; 103(4):318-24. PubMed ID: 17502272.
    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.
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