EGFP-based evaluation of temperature inducible native promoters of industrial ale yeast by using a high throughput system

• Characterizing of promoter activities under brewing conditions by using HTS.
• Evaluation of native promoters of industrial ale yeast in direct comparison.
• Temperature shift for maturation leads to substantial increase of gene expression.
• Induction effected by ethanol is dose dependent.
• PSSA3 have the strongest induction by shifts to cold temperature.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future requirements. Up to now, there is limited data of inducible expression patterns for targeted gene expression under such specific conditions. For the evaluation of temperature induced native promoters, the widely used reporter gene “enhanced green fluorescence protein” (EGFP) by utilizing high throughput systems was applied. Five different promoters of the industrial yeast strain Saccharomyces cerevisiae TUM 68 were evaluated (PHSP12, PHSP26, PHSP30, PHSP104, and PSSA3). They are induced during temperature shifts, which may occur in transition of fermentation to maturation. Furthermore, the induction of gene expression affected by different contents of ethanol were investigated, by using synthetic wort which mimics a 12 °P wort. Promoters PHSP30 and PSSA3 showed the highest fluorescence value during temperature shift from 20 °C to 10 °C. A temperature shift from 20 °C to 4 °C, resulted in highest fluorescence values of PSSA3 and PHSP26. Further, these promoters showed the lowest induction value by ethanol concentrations between 4 and 6%-vol. With this method, it is possible to evaluate native temperature induced promoters for the usage in self-cloning brewing yeast under strict industrial conditions.