Diacetyl causes an unwanted buttery off-flavor in lager beer. The production of diacetyl is reduced by modifying the metabolic pathway of yeast in the beer fermentation process. In this study, BDH2 and ILV5 genes, coding diacetyl reductase and acetohydroxy acid reductoisomerase, respectively, were expressed using a PGK1 promoter in Saccharomyces cerevisiae, which deleted one ILV2 allelic gene. Diacetyl contents and fermentation performances were examined and compared. Results showed that the diacetyl content in beer was remarkably reduced by 16.52% in QI2-KP (one ILV2 allelic gene deleted), 55.65% in QI2-B2Y (overexpressed BDH2 gene and one ILV2 allelic gene deleted), and 69.13% in QI2-I5Y (overexpressed ILV5 gene and one ILV2 allelic gene deleted) compared with the host strain S2. The fermentation ability of mutant strains was similar to that of S2. Results of the present study can lead to further advances in this technology and its broad application in scientific investigations and industrial beer production.

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

Diacetyl is crucial to beer maturation because of its characteristic buttery taste. New industrial brewer’s yeast strains, including single ILV2 allele disruption (QI2), single ILV2 allele disruption and BDH1 overexpression (QI2GB1), and BDH1 overexpression (B1Y), were constructed by disrupting the gene (ILV2) involved in a-acetolactate synthesis and introducing the diacetyl reductase gene (BDH1). The diacetyl content and fermentation performance of the three recombinant strains were examined and compared. Results showed that the diacetyl concentration in QI2, QI2GB1, and B1Y decreased by 17.83, 28.26, and 9.57 %, respectively, compared with that in the host strain S2. The fermentation ability in terms of fermentation degree and major flavors yield of the three recombinant strains was similar to that of the host. This study may serve as a reference for the brewing industry and for research on diacetyl reduction.

Baker’s yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker’s yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker’s yeast using the method proposed in this paper.

•The decrease in PrA activity can reduce the concentration of biogenic amines.•The production of biogenic amines decreased by 25.5% in Chinese rice wine.•This study is the first to show that the PrA activity of yeast affects the production of biogenic amines.Biogenic amines in Chinese rice wine have a potential threat of toxicity to human health. In this study, PEP4 gene in Saccharomyces cerevisiae was knocked out in order to evaluate its effect on biogenic amines production; the enzyme encodes proteinase A (PrA), an enzyme that is responsible for the production of free amino acids. It was found that compared to the wild type strain, the PrA activity and amino acid concentration decreased significantly, and the production of biogenic amines in this knockout strain decreased by 25.5%, from 180.1 mg/L to 134.2 mg/L. Especially, tyramine, cadaverine and histamine concentrations were also decreased by 57.5%, 24.6% and 54.3%, respectively. The main reason for the decrease of biogenic amines may be due to the low concentration of free amino acids. Our results provide a new strategy to minimize the biogenic amine production during fermentation of Chinese rice wine.

A method based on headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used for the analysis of volatile compounds in “Hengshui Laobaigan” liquor. Five different fibers were evaluated in terms of the number of volatile compounds, sensitivity and reproducibility. The results showed that 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was most suitable for acquiring a complete profile of the volatile compounds in Laobaigan liquor. For specific applications, 75 μm carboxen/polydimethylsiloxane (CAR/PDMS) fiber was appropriate to extract acids, alcohols, pyrazines and aromatic and phenolic compounds because of its high sensitivity, and 50/30 μm DVB/CAR/PDMS fiber was found to have higher sensitivity than others for extracting esters, hydrocarbons, aldehydes and ketones. It is concluded that different fibers should be selected depending on different research objects for acquiring accurate and reliable results.

Dough-leavening ability is one of the main aspects considered when selecting a baker’s yeast strain for baking industry. Generally, modification of maltose metabolic pathway and known regulatory networks of maltose metabolism were used to increase maltose metabolism to improve leavening ability in lean dough. In this study, we focus on the effects of PGM2 (encoding for the phosphoglucomutase) and SNR84 (encoding for the H/ACA snoRNA) that are not directly related to both the maltose metabolic pathway and known regulatory networks of maltose metabolism on the leavening ability of baker’s yeast in lean dough. The results show that the modifications on PGM2 and/or SNR84 are effective ways in improving leavening ability of baker’s yeast in lean dough. Deletion of PGM2 decreased cellular glucose-1-phosphate and overexpression of SNR84 increased the maltose permease activity. These changes resulted in 11, 19 and 21 % increases of the leavening ability for PGM2 deletion, SNR84 overexpression and SNR84 overexpression combining deleted PGM2, respectively.

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.

As the most important group in the flavor profiles of Chinese liquor, ester aroma chemicals are responsible for the highly desired fruity odors. Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis in Saccharomyces cerevisiae. In this study, we overexpressed ATF1 in Chinese liquor yeast through precise and seamless insertion of PGK1 promoter (PGK1p) via a novel fusion PCR-mediated strategy. After two-step integration, PGK1p was embedded in the 5′-terminal of ATF1 exactly without introduction of any extraneous DNA sequence. In the liquid fermentation of corn hydrolysate, both mRNA level and AATase activity of ATF1 in mutant were pronounced higher than the parental strain. Meanwhile, productivity of ethyl acetate increased from 25.04 to 78.76 mg/l. The self-cloning strain without any heterologous sequences residual in its genome would contribute to further commercialization of favorable organoleptic characteristics in Chinese liquor.

A method based on headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry was developed for the analysis of volatile methoxyphenolic compounds in pu-erh tea. Six fibers with different polarities were initially evaluated. The 75 μm carboxen/polydimethylsiloxane fiber exhibited the highest extraction efficiency and was selected for further optimization. A Plackett–Burman design was used to screen for the brewing proportion of tea and water, amount of pu-erh tea, ionic strength, extraction time, extraction temperature, desorption time, rate of agitation, and equilibrium time. A Box–Behnken design was then applied to optimize the significant factors. Under optimal conditions, the proposed method affords a wide range of linearity, high linear regression coefficients (0.996–0.999), less than 9.0% repeatability of relative standard deviation, and limits of detection ranging from 2.31 to 21.80 ng/g. The proposed method has satisfactory accuracy, with recoveries of 79.08–113.9%. This method was successfully applied for the analysis of pu-erh tea samples.

Esters and higher alcohols produced by yeast during the fermentation of wort have the greatest impact on the smell and taste of beer. Alcohol acetyltransferase, which is mainly encoded by the ATF1 gene, is one of the most important enzymes for acetate ester synthesis. Cytosolic branched-chain amino acid aminotransferase, on the other hand, which is encoded by the BAT2 gene, plays an important role in the production of branched-chain alcohols. The objective of this study is to construct engineered brewer’s yeast strains that produce more acetate esters and less higher alcohols. Industrial brewer’s yeast strain S5 was used as the parental strain to construct ATF1 overexpression and BAT2 deletion mutants. The engineered strains S5-2 and S5-4, which feature partial BAT2 allelic genes replaced by the constructed ATF1 overexpression cassette, were obtained. The ester production of the engineered strains was observed to increase significantly compared with that of the parental cells. The concentrations of ethyl acetate produced by the engineered strains S5-2 and S5-4 increased to 78.88 and 117.40 mg L−1, respectively, or about 7.7-fold and 11.5-fold higher than that produced by parental S5 cells. The isoamyl acetate produced by S5-2 and S5-4 also increased to 5.14 and 9.25 mg L−1, respectively; by contrast, no isoamyl acetate was detected in the fermentation sample of the parental strain S5. Moreover, S5-2 and S5-4, respectively, produced about 65 and 51 % of higher alcohols produced by the parental strain. The increase in acetate ester content and decrease in higher alcohol concentration shown by the engineered brewer’s yeast strains at the end of fermentation process indicate that the new strains are useful in future developments in the wheat beer industry.

Two lactose-consuming diploid Saccharomyces cerevisiae strains, AY-51024A and AY-51024M, were constructed by expressing the LAC4 and LAC12 genes of Kluyveromyces marxianus in the host strain AY-5. In AY-51024A, both genes were targeted to the ATH1 and NTH1 gene-encoding regions to abolish the activity of acid/neutral trehalase. In AY-51024M, both genes were respectively integrated into the MIG1 and NTH1 gene-encoding regions to relieve glucose repression. Physiologic studies of the two transformants under anaerobic cultivations in glucose and galactose media indicated that the expression of both LAC genes did not physiologically burden the cells, except for AY-51024A in glucose medium. Galactose consumption was initiated at higher glucose concentrations in the MIG1 deletion strain AY-51024M than in the corresponding wild-type strain and AY-51024A, wherein galactose was consumed until glucose was completely depleted in the mixture. In lactose medium, the Sp. growth rates of AY-51024A and AY-51024M under anaerobic shake-flasks were 0.025 and 0.067 h−1, respectively. The specific lactose uptake rate and ethanol production of AY-51024M were 2.50 g lactose g CDW−1 h−1 and 23.4 g l−1, respectively, whereas those of AY-51024A were 0.98 g lactose g CDW−1 h−1 and 24.3 g lactose g CDW−1 h−1, respectively. In concentrated cheese whey powder solutions, AY-51024M produced 63.3 g l−1 ethanol from approximately 150 g l−1 initial lactose in 120 h, conversely, AY-51024A consumed 63.7 % of the initial lactose and produced 35.9 g l−1 ethanol. Therefore, relieving glucose repression is an effective strategy for constructing lactose-consuming S. cerevisiae.

This study aimed to increase maltose fermentation in industrial baker’s yeast to increase its leavening properties. To this end, we overexpressed MAL62 encoding alpha-glucosidase (maltase) and deleted MIG1 encoding a transcriptional repressor that regulates MAL gene expression. Strain overexpressing MAL62 showed 46.3 % higher alpha-glucosidase activity and enhanced leaving activity than the parental strain when tested in glucose–maltose low sugar model liquid dough (LSMLD). Deleting MIG1 was much less effective, but it could further strengthen leavening properties in a strain overexpressing MAL62. The relationship between maltose permease and alpha-glucosidase was further dissected by transforming the two genes. The results indicated that without increasing the maltose permease activity, maltose metabolism could also be enhanced by the increased alpha-glucosidase activity. Previous strategies for strain improvement have targeted the enhancement of alpha-glucosidase and maltose permease activities in concert. Our results suggest that increasing alpha-glucosidase activity is sufficient to improve maltose fermentation in lean dough.

Baker’s yeast,Saccharomyces cerevisiae, is a key microorganism used in the baking industry. While the preferred substrate for baker’s yeast is generally glucose, the predominant carbohydrate in lean dough is maltose. Therefore, in order to improve the leavening properties of lean dough, it is essential to improve maltose metabolism by the yeast. The objective of this study was to gain better insight into the regulation of the yeast maltose-transporter, maltose permease, and the maltose-cleaving enzyme, maltase, by glucose in lagging and non-lagging strains of baker’s yeast. Gas evolution in a low sugar model liquid dough (LSMLD) medium was used to select five out of ten industrial baker’s yeast strains for further investigation on the basis of varying metabolic characteristics. In all four of the lagging strains tested, both maltose permease and maltase were inhibited by glucose to some extent. In the relative non-lagging strain, which demonstrated the highest performance in LSMLD, it was shown that maltase was not inhibited by glucose. Based on our findings, it indicated that in lean dough leavening, it is the maltase that plays the essential role in maltose metabolism, rather than the maltose permease. Therefore, we propose that the lack of glucose repression on maltase activity is the most critical criterion in the development of non-lagging strains of baker’s yeast.