Development of Stable Flocculent Saccharomyces cerevisiae Strain for Continuous Aspergillus niger β-Galactosidase Production

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.