Functional enhancement of Sake yeast strains to minimize the production of ethyl carbamate in Sake wine

Abstract

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.

Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

“…There were two aspects to the experiments using the model rice wine system. On one hand, the effect of the UG strain on EC elimination was far superior to the effects of the other methods (9,10,32,33). On the other hand, the concentrations of the main components in rice wine changed very little (all of the changes were Ͻ5%).…”

Improving industrial yeast strains: exploiting natural and artificial diversity

“…In aging wines or sake, the concentration of ethyl carbamate, which is mainly formed by a spontaneous reaction of ethanol with urea, can be relatively high (Pretorius & Bauer, 2002; Dahabieh et al ., 2010). By adjusting the urea catabolism of the yeast strain, ethyl carbamate concentrations can be significantly lowered (Kitamoto et al ., 1991; Coulon et al ., 2006; Dahabieh et al ., 2010). The most successful resulting strain so far, called 522 EC− , was approved for commercial use (Chambers & Pretorius, 2010).…”

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression ofDUR3for ethyl carbamate elimination

“…Furthermore, it is hypothesized that more copies of DUR3 combined with the DUR1,2 expression cassette in the yeast cell could be even more effective in reducing the urea and EC concentration. Different from the previous study, which also integrated PKG1p in the upstream of DUR3 gene, in the present study no antibiotic genes were introduced, but there was a higher exhibition of the DUR3 expression level and the yeast was more effective in reducing the urea concentration and EC formation (8). Thus, the genetically modified strains from this current study could have a good industrial application potential, since no exogenous genes or antibiotic genes were introduced during the genetic manipulation process.…”

“…Urea can be imported into a S. cerevisiae cell in two ways. One route is active transport through the membrane using urea permease, which is encoded by the NCR sensitive DUR3 gene, and the other route is via either passive or facilitated diffusion, when the extracellular urea concentration is 0.5 mmol L À1 or above (6)(7)(8).…”