A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

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 ß-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.

Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.