The phenotypic characterization of yeast strains to stresses inherent to wine fermentation in warm climates.

AIMS: Climate change is exerting an increasingly profound effect on grape composition, microbiology, chemistry and the sensory aspects of wine. Identification of autochthonous yeasts tolerant to stress could help to alleviate this effect. METHODS AND RESULTS: Tolerance to osmotic pressure, ethanol and pH of 94 Saccharomyces cerevisiae strains and 29 strains non-Saccharomyces from the warm climate region DO 'Vinos de Madrid' (Spain) using phenotypic microarray and their fermentative behaviour were studied. The screening highlighted 12 strains of S. cerevisiae isolated from organic cellars with improved tolerance to osmotic stress, high ethanol concentrations and suitable fermentative properties. Screening of non-Saccharomyces spp. such as Lanchacea thermotolerans, Torulaspora delbrueckii, Schizosaccharomyces pombe and Mestchnikowia pulcherrima also highlighted tolerance to these stress conditions. CONCLUSIONS: This study confirmed the adaptation of native strains to the climatic conditions in each area of production and correlated these adaptations with good fermentation properties. Screening has revealed that identifying yeast strains adapted to fermentation stresses is an important approach for making quality wines in very warm areas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have special relevance because it is a pioneering study that has approached the problem of climate change for wines from a microbiological aspect and has analysed the situation in situ in wineries from a warm climate zone. Resistant strains were found with good biological properties; studying these strains for their stress response mechanisms during fermentation will be of interest to the wine making industry.

Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress.

BACKGROUND: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars during beverage or bioethanol fermentations. These fermentations are characterised by high osmotic stress on a yeast cell, with selected brewing fermentations beginning at 20-25% fermentable sugars and bioethanol fermentations at 13% fermentable sugars. RESULTS: RCK2 encodes for a MAPKAP (MAPK-activated protein kinase) enzyme and was identified on a locus by QTL analysis in yeast cells under osmotic stress, RCK2 expression was placed under a tetracycline regulatable vector and rescued glucose, sorbitol or glycerol induced osmotic stress in an rck2 null strain. A strain overexpressing RCK2 had significantly faster fermentation rates when compared with the empty vector control strain. CONCLUSIONS: Presence of RCK2 increased rates of glucose utilisation (~40 g glucose in first 8 h) during a 15% glucose fermentation and concurrent production of ethanol when compared with empty vector controls. Tolerance to osmotic stress using the tetracycline regulatable vectors could be turned off with the addition of tetracycline returning a rck2 null strain back to osmotic sensitivity.

Phenotypic characterisation of Saccharomyces spp. for tolerance to 1-butanol.

Biofuels are expected to play a role in replacing crude oil as a liquid transportation fuel, and research into butanol has highlighted the importance of this alcohol as a fuel. Butanol has a higher energy density than ethanol, butanol-gasoline blends do not separate in the presence of water, and butanol is miscible with gasoline (Szulczyk, Int J Energy Environ 1(1):2876-2895, 40). Saccharomyces cerevisiae has been used as a fermentative organism in the biofuel industry producing ethanol from glucose derived from starchy plant material; however, it typically cannot tolerate butanol concentrations greater than 2 % (Luong, Biotechnol Bioeng 29 (2):242-248, 27). 90 Saccharomyces spp. strains were screened for tolerance to 1-butanol via a phenotypic microarray assay and we observed significant variation in response with the most tolerant strains (S. cerevisiae DBVPG1788, S. cerevisiae DBVPG6044 and S. cerevisiae YPS128) exhibiting tolerance to 4 % 1-butanol compared with S. uvarum and S. castelli strains, which were sensitive to 3 % 1-butanol. Response to butanol was confirmed using traditional yeast methodologies such as growth; it was observed that fermentations in the presence of butanol, when using strains with a tolerant background, were significantly faster. Assessing for genetic rationale for tolerance, it was observed that 1-butanol-tolerant strains, when compared with 1-butanol-sensitive strains, had an up-regulation of RPN4, a transcription factor which regulates proteasome genes. Analysing for the importance of RPN4, we observed that a Δrpn4 strain displayed a reduced rate of fermentation in the presence of 1-butanol when compared with the BY4741 background strain. This data will aid the development of breeding programmes to produce better strains for future bio-butanol production.