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Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85 DUR1,2/DUR3 -c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85 DUR1,2/DUR3 -c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Ethyl carbamate (EC) is a potentially carcinogenic substance present in most alcoholic beverages, especially in Chinese rice wine. Consequently, much effort has been directed at suppressing EC formation during the production of these beverages, with particular attention directed at the use of urethanase, as this enzyme can directly catalyze EC degradation. Herein, we investigated the ability of three lactic acid bacteria (Oenococcus oeni, Lactobacillus brevis, and Lactobacillus plantarum) to generate urethanase during co-cultivation with Saccharomyces cerevisiae. qPCR and transcriptomic analyses revealed that 57 genes of S. cerevisiae were significantly expressed in the presence of L. brevis, which highlighted the importance of studying urethanase-promoted EC degradation for establishing a powerful technique of EC level control. The obtained results provided deep insights into the adaptive responses of S. cerevisiae to the challenging environment of mixed-culture fermentation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Producing alcoholic beverages with novel flavor are desirable for winemakers. We created fermenting yeast with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Strategies of ethanol domestication, ultraviolet mutagenesis (UV) and protoplast fusion were conducted to create yeast hybrids with excellent oenological characteristic. The obtained diploid hybrid F23 showed a cell viability of 6.2% under 25% ethanol, whereas its diploid parental strains could not survive under 20% ethanol. During Chinese rice wine-making, compared to diploid parents, F23 produced 7.07%-12.44% higher yield of ethanol. Flavor analysis indicated that the total content of flavor compounds in F23 wine was 19.99-26.55% higher than that of parent wines. Specifically, F23 exhibited higher capacity in producing 2-phenylethanol, short-chain and long-chain fatty-acid ethyl-ester than diploid parents. Compared to diploid parents, F23 introduced more flavor contributors with odor activity values (OAVs) above one to Chinese rice wine, and those contributors were found with higher OAVs. Based on principal component analysis (PCA), the flavor characteristic of F23 wine was similar to each of parent wine. Additionally, sensory evaluation showed that F23 wine was highly assessed for its intensive levels in fruit-aroma, alcohol-aroma and mouthfeel. Hybrid F23 not only displayed superior flavor production and oenological performance in making Chinese rice wine, but also could act as potential "mixed-like" starter to enrich wine style and differentiation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Saccharomyces cerevisiae metabolizes arginine to ornithine and urea during wine fermentations. In the fermentation of Chinese rice wine, yeast strains of S. cerevisiae do not fully metabolize urea, which will be secreted into the spirits and spontaneously reacts with ethanol to form ethyl carbamate, a potential carcinogenic agent for humans. To block the pathway of urea production, we genetically engineered two haploid strains to reduce the arginase (encoded by CAR1) activity, which were isolated from a diploid industrial Chinese rice wine strain. Finally the engineered haploids with opposite mating type were mated back to diploid strains, obtaining a heterozygous deletion strain (CAR1/car1) and a homozygous defect strain (car1/car1). These strains were compared to the parental industrial yeast strain in Chinese rice wine fermentations and spirit production. The strain with the homozygous CAR1 deletion showed significant reductions of urea and EC in the final spirits in comparison to the parental strain, with the concentration reductions by 86.9% and 50.5% respectively. In addition, EC accumulation was in a much lower tempo during rice wine storage. Moreover, the growth behavior and fermentation characteristics of the engineered diploid strain were similar to the parental strain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To investigate the effect of deleting the genes encoding branched-chain amino acid transaminase (BAT2 gene) and pyruvate decarboxylase-like enzyme (THI3 gene) in Saccharomyces cerevisiae on the high alcohol content in Chinese rice wine, the diploid yeasts XF1-B (BAT2 gene deletion) and XF1-TB (THI3 and BAT2 gene deletion) were constructed using the Cre/loxP recombination system with the haploid XF1a7/XF1α6 of XF1 and haploid XF1a7-T/XF1α6-T of XF1-T (THI3 gene deletion) as the original strains. Chinese rice wine was fermented using XF1, XF1-B, and XF1-TB under the same conditions. The growth capability of XF1-B and XF1-TB was similar to that of XF1, and the basic fermentation capability was not significantly different. The isoamyl alcohol content decreased by 18.12% and 26.06%, the isobutanol content decreased by 35.21%and 18.83%, the n-propanol content increased by 15.42%and 32.75%, and the high alcohol content decreased by 15.65%and 15.28%to 269.59 and 270.77 mg/L in the Chinese rice wine brewed using XF1-B and XF1-TB, respectively, compared with those of wine brewed using XF1. The ratio of isoamyl alcohol/isobutanol was 3.24 and 2.34 in Chinese rice wine brewed using XF1-B and XF1-TB, respectively, representing a decrease of 27.78% compared with that of wine brewed using XF1. In summary, both XF1-B and XF1-TB effectively reduced the total higher alcohol content, and XF1-TB effectively reduced the ratio of isoamyl alcohol/isobutanol in Chinese rice wine. Hence, they can weaken the intoxication degree and improve the quality of Chinese rice wine [ABSTRACT FROM AUTHOR]

Abstract: Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85ARG1,4 and N85ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85ARG1,4 and N85ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

The volatile compounds of Chinese rice wine fermented by Saccharomyces cerevisiae FC 15 and S. cerevisiae BR 30 and their combination (MIX) were investigated. The results showed that the wines fermented with different starters differed mainly in the numbers of alcohols and esters. Furthermore, the amounts of volatile compounds among three starters were also obviously different. After clarification, the loss of esters was greater than that of alcohols and aldehydes, whereas after sterilization, the loss of aldehydes was the greatest. PCA showed diethyl succinate to be the key flavor component. Moreover, short-chain fatty acid ethyl esters were main flavor substances during fermentation, whereas long-chain fatty acid ethyl esters were typical compounds after clarification and sterilization. Analysis of the ratio of higher alcohols to esters showed that BR 30 wine presented a stronger ester aroma than MIX wine, whereas FC 15 wine exhibited an outstanding alcohol flavor. Sensory evaluation indicated that FC 15 wine was intense in alcohol-aroma, whereas BR 30 wine exhibited high levels of cereal-aroma. Furthermore, both MIX and BR 30 wine were highly assessed in continuation and full body mouth-feel. It means that flavor characteristic of Chinese rice wine could be adjust by combination of yeast starters. [ABSTRACT FROM AUTHOR]

In recent years, potential probiotic strains have been increasingly applied to food to promote consumer health or improve the nutrition of products. In current study, a potential probiotic Saccharomyces cerevisiae strain BR14 was applied for the development of functional Huangjiu. The strain significantly increased the ethanol content in final wine, as well as the total acidity, especially acetic acid and lactic acid. As an accessory starter, BR14 promoted the formation of total volatile flavor compounds, particularly in 1-propanol, 3-methyl-1-butanol and phenethyl alcohol. Meanwhile, it reduced the formation of 2-Methyl-1-propanol by 54.65%. Regarding nutrients and functionality of final Chinese rice wine, S. cerevisiae BR14 exhibited excellent capacity in the improvement of glutamic acid, ornithine and antiradical activity for wine production. Content of urea, which determined the formation of ethyl carbamate, considerably decreased by the co-fermentation of BR14. Moreover, polysaccharide of Huangjiu fermented by BR14 stimulated the proliferation of macrophages RAW264.7, and reduced anti-inflammatory response. Based on sensory evaluation, BR14 wine was highly assessed for its intensive levels in alcohol-aroma and full body. This study verified the biofunctions of BR14, indicating the possibility to apply potential probiotic yeasts in food applications. • A potential probiotic yeast strain BR14 was used to develop functional Huangjiu. • BR14 greatly contributed to the improvement of organic acids and aromatic alcohols. • BR14 reduced the content of urea and 2-methyl-1-propanol. • BR14 enhanced content of non-essential amino acids (Glu and Orn). • BR14 improved the healthy characteristics of rice wine. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the combined effect of thermosonication (TS) and pulsed electric fields (PEF) against Saccharomyces cerevisiae in Chinese rice wine. The effectiveness of standalone TS treatment (35 °C, 750 W, 120 min) on the inactivation of S. cerevisiae was insignificant (0.76 log CFU/mL). However, 2.88 log CFU/mL of S. cerevisiae were inactivated when the standalone PEF treatment with moderate conditions (35 °C, 12 kV/cm, 120 μs) was applied. The combination of TS and PEF had an additive effect on the inactivation of S. cerevisiae, and the sequence applied (TS-PEF or PEF-TS) markedly influenced the inactivation results ( P < 0.05). In particular, the microbial inactivation by TS-PEF (3.72 log CFU/mL) was higher than that by PEF-TS (3.48 log CFU/mL); this result indicates that PEF were able to restrain the effect of TS. On the other hand, TEM micrographs of S. cerevisiae after the different treatments showed that the combined techniques resulted in more severe disruptions on cells. Higher cytoplasmic shrinkage and more intracellular material leakage were observed from the TEM observations of the cells treated by TS-PEF. These results may serve as a reference of the potential application of the combined treatment TS-PEF for microbial inactivation in Chinese rice wine. [ABSTRACT FROM AUTHOR]

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small-scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non-preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT-PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85DUR1,2 and N85DUR1,2-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85DUR1,2 and N85DUR1,2-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine. [ABSTRACT FROM AUTHOR]

An appropriate level of higher alcohols produced by yeast during the fermentation is one of the most important factors influencing Chinese rice wine quality. In this study, BAT1 and BAT2 single- and double-gene-deletion mutant strains were constructed from an industrial yeast strain RY1 to decrease higher alcohols during Chinese rice wine fermentation. The results showed that the BAT2 single-gene-deletion mutant strain produced best improvement in the production of higher alcohols while remaining showed normal growth and fermentation characteristics. Furthermore, a BAT2 single-gene-deletion diploid engineered strain RY1-Δ bat2 was constructed and produced low levels of isobutanol and isoamylol (isoamyl alcohol and active amyl alcohol) in simulated fermentation of Chinese rice wine, 92.40 and 303.31 mg/L, respectively, which were 33.00 and 14.20 % lower than those of the parental strain RY1. The differences in fermentation performance between RY1-Δ bat2 and RY1 were minor. Therefore, construction of this yeast strain is important in future development in Chinese wine industry and provides insights on generating yeast strains for other fermented alcoholic beverages. [ABSTRACT FROM AUTHOR]

High tolerance towards ethanol is a desirable property for the Saccharomyces cerevisiae strains used in the alcoholic beverage industry. To improve the ethanol tolerance of an industrial Chinese rice wine yeast, a sequential batch fermentation strategy was used to adaptively evolve a chemically mutagenized Chinese rice wine G85 strain. The high level of ethanol produced under Chinese rice wine-like fermentation conditions was used as the selective pressure. After adaptive evolution of approximately 200 generations, mutant G85X-8 was isolated and shown to have markedly increased ethanol tolerance. The evolved strain also showed higher osmotic and temperature tolerances than the parental strain. Laboratory Chinese rice wine fermentation showed that the evolved G85X-8 strain was able to catabolize sugars more completely than the parental G85 strain. A higher level of yeast cell activity was found in the fermentation mash produced by the evolved strain, but the aroma profiles were similar between the evolved and parental strains. The improved ethanol tolerance in the evolved strain might be ascribed to the altered fatty acids composition of the cell membrane and higher intracellular trehalose concentrations. These results suggest that adaptive evolution is an efficient approach for the non-recombinant modification of industrial yeast strains. [ABSTRACT FROM AUTHOR]

The application of pulsed electric field (PEF) technology as an alternative method of spoilage microbiological control for Chinese rice wine was investigated. PEF was applied at the initial temperature of 25–35°C, the electric field strength of 12–21kV/cm, with respective treatment time of 30–180μs to evaluate the efficiency of this technology on the inactivation of spoilage yeast Saccharomyces cerevisiae commonly associated with rice wine. The results from atomic force microscope imaging of yeast cells indicated that PEF treatment induced the destruction of cell membrane structures, which supported by the decrease of yeast number, the blurring of images, and the flattened yeast border. The highest inactivation values, corresponding to approximately 5.5-log cycles, were obtained at 21kV/cm and 180μs with the initial treatment temperatures of 30 and 35°C, respectively. Based on calculations by both log-linear and Weibull models, the decimal reduction times (5-D values) over the range of treatment conditions were 336.53–1804.1μs and 150.17–1127.6μs, respectively. Then the 5-D values were fitted as a function of electric field strength and initial treatment temperature, with R 2 of 0.986 and 0.963 for log-linear and Weibull model, respectively. The model validation results showed that the 5-D values calculated from Weibull model (Af =1.169 and Bf =0.996) provided more accuracy than those from log-linear model (Af =1.773 and Bf =1.773), suggesting that the Weibull model was more suitable for calculating the survival ratio of PEF-treated spoilage microorganisms in Chinese rice wine. Finally, the effects of PEF treatment on physicochemical properties (including °Brix, pH, conductivity and color) of rice wine exactly after treatment and during storage at room temperature (22°C) were also investigated. The results indicated that PEF was able to inactivate (to some degree) the yeast in rice wine with minor variations in the physicochemical parameters and affect the quality indices of rice wine positively. [ABSTRACT FROM AUTHOR]