Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger ¦Â-galactosidase production

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• 作者：Carla  ; Oliveira^a ; José ; A.  ; Teixeira^a ; Nelson  ; Lima^a ; Nancy A.  ; Da Silva^b ; Lucí ; lia  ; Domingues^a ;   ; ^{luciliad@deb.uminho.pt}
• 关键词：genetic stability of delta-integrating systems ; continuous high-cell-density culture ; Aspergillus niger ¦Â-galactosidase production ; recombinant ; Saccharomyces cerevisiae ; yeast flocculation
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2007
• 出版时间：April 2007
• 年：2007
• 卷：103
• 期：4
• 页码：318-324
• 全文大小：362 K

文摘

A flocculent Saccharomyces cerevisiae strain was engineered to stably secrete Aspergillus niger ¦Â-galactosidase in a continuous high-cell-density bioreactor. The ¦Ä-sequences from the yeast retrotransposon Ty1 were used as target sites for the integration of the ¦Â-galactosidase expression cassette. High-copy-number transformants were successfully obtained using the ¦Ä-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 ¦Â-galactosidase levels (approximately eight gene copies) had similar ¦Â-galactosidase activities as a recombinant strain carrying the ¦Â-galactosidase expression cassette in a YEp-based vector. The ¦Â-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 ¦Ä-integrant stability in a continuous bioreactor operating at different dilution rates.