Biomarkers for detecting nitrogen deficiency during alcoholic fermentation in different commercial wine yeast strains

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Several putative biomarkers were tested in order to analyze their appropriateness to detect nitrogen stress in the yeast. To this aim, four commercial wine strains (PDM, ARM, RVA and TTA) were grown in a synthetic grape must with different nitrogen concentrations. Trehalose accumulation, arginase activity and the expression of eleven genes were tested in these wine strains, known to have different nitrogen requirements. The overall response of the four strains was similar, with differences in response intensity (PDM and RVA with higher intensity) and response time (which was also related with nitrogen consumption time). Trehalose response was mostly related to entry into the stationary phase, whereas arginase activity was responsive to nitrogen depletion, although its measurement is too complicated to be used for routine monitoring during winemaking. The expression of the genes DAL4, DAL5, DUR3 and GAP1 was clearly related to nitrogen depletion and thus, GAP1 and DAL4 were selected as markers of nitrogen deficiency. In order to adapt expression analysis to winemaking conditions, the original strains were transformed into reporter strains based on the expression of green fluorescent protein (GFP) under control of the promoters for GAP1 and DAL4. The transformants had a similar fermentative capacity to the parental strains and were able to detect alterations in yeast physiological status due to nitrogen limitations.