First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation.

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl-CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re-oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.

Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.