Oliveira Carla, Teixeira José A, Lima Nelson, Da Silva Nancy A, Domingues Lucília
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
J Biosci Bioeng. 2007 Apr;103(4):318-24. doi: 10.1263/jbb.103.318.
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.
构建了一种絮凝型酿酒酵母菌株，使其在连续高细胞密度生物反应器中稳定分泌黑曲霉β-半乳糖苷酶。酵母逆转座子Ty1的δ序列用作β-半乳糖苷酶表达盒整合的靶位点。使用δ整合系统和显性选择抗生素G418成功获得了高拷贝数转化体。通过基因组Southern印迹分析证实了多拷贝的整合。β-半乳糖苷酶水平最高（约八个基因拷贝）的整合体具有与在基于YEp的载体中携带β-半乳糖苷酶表达盒的重组菌株相似的β-半乳糖苷酶活性。在非选择性培养基中进行八次连续分批培养后，整合到酵母基因组中的β-半乳糖苷酶表达盒得以稳定维持。在相同操作条件下的连续高细胞密度培养中，整合体菌株比携带质粒的菌株更稳定。据我们所知，这是首次在以不同稀释率运行的连续生物反应器中研究多拷贝δ整合体稳定性的研究。
