A New Expression System Based on Psychrotolerant <i>Debaryomyces macquariensis</i> Yeast and Its Application to the Production of Cold-Active β-<span style="font-variant: small-caps">d</span>-Galactosidase from <i>Paracoccus</i> sp. 32d

Abstract

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 Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis 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 GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis 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.

Keywords

• <i>Debaryomyces macquariensis</i> strain D50
• psychrotolerant yeast
• host/vector expression system
• constitutive <i>GAP</i> promoter
• constitutive <i>TEF1</i> promoter
• <i>CYC1</i> transcriptional terminator