Development of Stable Flocculent Saccharomyces cerevisiae Strain for Continuous Aspergillus niger β-Galactosidase Production(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)

概要

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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 Tyl 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.
公益社団法人日本生物工学会の論文
2007-04-25