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

Carla Oliveira, José A. Teixeira, Nelson Lima, Nancy A. da Silva, Lucília Domingues*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)318-324
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume103
Issue number4
DOIs
Publication statusPublished - Apr 2007
Externally publishedYes

Keywords

  • Aspergillus niger β-galactosidase production
  • continuous high-cell-density culture
  • genetic stability of delta-integrating systems
  • recombinant
  • Saccharomyces cerevisiae
  • yeast flocculation

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