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Aims:  In fermented alcoholic beverages and particularly in Japanese Sake wine, the ubiquitous presence of the probable human carcinogen ethyl carbamate (EC) is a topic of significant concern. This study aims to develop novel methods for the reduction of EC in Sake wine. Methods and Results:  To reduce the high levels of EC in Sake wine, urea‐degrading and urea‐importing yeast strains were created by integrating linear cassettes containing either the respective DUR1,2 or DUR3 genes, under the control of the constitutively active Saccharomyces cerevisiae PGK1 promoter, into the Sake yeast strains K7 and K9. The self‐cloned, urea‐degrading Sake strains K7DUR1,2 and K9DUR1,2 produced Sake wine with 87 and 68% less EC, respectively, while the urea‐importing Sake yeast strain K7DUR3 reduced EC by 15%. All functionally enhanced yeast strains were shown to be substantially equivalent to their parental strains in terms of fermentation rate, ethanol production, phenotype and transcriptome. Conclusions:  Under the conditions tested, urea‐degrading yeast (constitutive DUR1,2 expression) are superior to urea‐importing yeast (constitutive DUR3 expression) for EC reduction in Sake wine, and constitutive co‐expression of DUR1,2 and DUR3 does not yield synergistic EC reduction. Significance and Impact of the Study:  The self‐cloned, substantially equivalent, urea‐degrading Sake yeast strains K7DUR1,2 and K9DUR1,2, which contain the integrated DUR1,2 cassette, are capable of highly efficacious EC reduction during Sake brewing trials, are suitable for commercialization and are important tools for modern Sake makers in their efforts to reduce high EC levels in Sake wine.

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The optimal ratio of L-malic and L-tartaric acid in relation to other wine components is one of the most important aspects that ultimately determine wine quality during winemaking. Winemakers routinely rely on the judicious use of malolactic fermentation (MLF) after alcoholic fermentation to deacidify and stabilise their wines. However, due to the unreliability of the process and unsuitable sensory modifications in some grape cultivars, especially for fruity-floral wines, MLF is often regarded as problematic and undesirable. Alternative methods for reducing the amounts of L-malic acid in wine will contribute to improving the production of quality wines in the future, especially in coolclimate regions. Most wine yeast strains of Saccharomyces are unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of malo-ethanolic fermentation. However, strains of S. pombe are not suitable for vinification due to the production of undesirable off-flavours. Previously, the 5. pombe malate permease (mael) and malic enzyme (mae2) genes were successfully expressed under the 3-phosphoglycerate kinase (PGK1) regulatory elements in 5. cerevisiae, resulting in a recombinant laboratory strain of S. cerevisiae with an efficient malo-ethanolic pathway. Stable integration of the S. pombe malo-ethanolic pathway genes has now been obtained through the construction of a unique integration strategy in a commercial wine yeast strain. Co-transformation of the linear integration cassette containing the mael and mae2 genes and PGK1 regulatory elements and a multi-copy plasmid containing the phleomycin-resistance marker into a commercial Saccharomyces cerevisiae strain resulted in the successful transformation and integration of the malo-ethanolic genes. The recombinant 5. cerevisiae strain was successfully cured of phleomycin-resistance plasmid DNA in order to obtain malo-ethanolic yeast containing only yeast-derived DNA. The integrated malo-ethanolic genes were stable in 5. cerevisiae and during synthetic and grape must fermentation, L-malic acid was completely fermented to ethanol without any negative effect on fermentation kinetics and wine quality.

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