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Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast <i>Debaryomyces</i> <i>macquariensis</i> strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for <i>D. macquariensis</i> D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive <i>GAP</i> promoter and <i>CYC1</i> transcriptional terminator from <i>D. macquariensis</i> D50 were constructed and used to clone and express a gene-encoding cold-active β-<span style="font-variant: small-caps;">d</span>-galactosidase of <i>Paracoccus</i> sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant <i>D. macquariensis</i> D50 strains were mitotically stable under both selective and non-selective conditions. The <i>D. macquariensis</i> D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.