CAR1 as a new selective marker for genetic engineering of wine yeasts.

A common problem in engineering industrial yeasts, and wine yeasts in particular, is the lack or scarcity of selective markers for introducing desired genetic changes. Almost all such markers, which are usually auxotrophic mutations, would reduce the growth characteristics of yeast strains. However, a potentially useful marker could be the CAR1 gene encoding arginase, the deletion of which reduces the accumulation of the carcinogen ethyl carbamate in wine, making such a deletion beneficial for wine production and maintainable in wine yeast strains. Here we demonstrate the use of the CAR1 gene as a selective marker. First, we observe that complete deletion of CAR1 in a triploid wine strain of Saccharomyces cerevisiae causes strong growth inhibition on a medium containing arginine as the only nitrogen source. Then, we show that strains with CAR1 deletion can be reliably transformed using CAR1 as a plasmid marker. Thus, the CAR1 gene can be used as a convenient selective marker in genetic engineering of wine yeasts, in particular using CRISPR/Cas9 technology.

Effects of pH and temperature on (S)-amine activity of transaminase from the cold-adapted bacterium Psychrobacter cryohalolentis.

(7R,8S)-diaminopelargonic acid transaminase from the cold-adapted Gram-negative bacterium Psychrobacter cryohalolentis (Pcryo361) is able to react with unnatural substrates including (S)-( +)-1-phenylethylamine, aldehydes and α-diketones. Additionally, Pcryo361 is active at 0-50 °C and retains up to 10% of the maximum activity at 0 °C. Here, we report a detailed study on the stability and low temperature activity of Pcryo361. At the optimal pH for (S)-amine activity (pH 10.0), the enzyme was stable at 0-10 °C and no decrease in the enzyme activity was observed within 24 h in a slightly alkaline medium, pH 8.0, at 35 °C. Pcryo361 was solvent stable and was activated in 10% DMSO and DMFA at 35 °C. An analysis of the efficiency of catalysis of Pcryo361 at 35 °C and 10 °C showed that the specificity towards (S)-( +)-1-phenylethylamine dropped at 10 °C; however, the specificity towards 2,3-butanedione remained unchanged. Inhibition analysis showed that Pcryo361 activity was not inhibited by acetophenone but inhibited by amines (products of aldehyde amination). The observed pH stability and low temperature activity of Pcryo361 with activated keto substrates are attractive features in the field of development of stereoselective amination at low temperatures.