Isolation and characterization of yeast for the production of rice wine with low fusel alcohol content
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ABSTRACT:
SUBMITTER: Yuan H
PROVIDER: S-EPMC8598244 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Branched-chain higher alcohols (BCHAs), or fusel alcohols, including isobutanol, isoamyl alcohol, and active amyl alcohol, are useful compounds in several industries. The yeast Saccharomyces cerevisiae can synthesize these compounds via the metabolic pathways of branched-chain amino acids (BCAAs). Branched-chain amino acid aminotransaminases (BCATs) are the key enzymes for BCHA production via the Ehrlich pathway of BCAAs. BCATs catalyze a bidirectional transamination reaction between branched-chain α-keto acids (BCKAs) and BCAAs. In S. cerevisiae, there are two BCAT isoforms, Bat1 and Bat2, which are encoded by the genes BAT1 and BAT2. Although many studies have shown the effects of deletion or overexpression of BAT1 and BAT2 on BCHA production, there have been no reports on the enhancement of BCHA production by functional variants of BCATs. Here, to improve BCHA productivity, we designed variants of Bat1 and Bat2 with altered enzyme activity by using in silico computational analysis: the Gly333Ser and Gly333Trp Bat1 and corresponding Gly316Ser and Gly316Trp Bat2 variants, respectively. When expressed in S. cerevisiae cells, most of these variants caused a growth defect in minimal medium. Interestingly, the Gly333Trp Bat1 and Gly316Ser Bat2 variants achieved 18.7-fold and 17.4-fold increases in isobutanol above that for the wild-type enzyme, respectively. The enzyme assay revealed that the catalytic activities of all four BCAT variants were lower than that of the wild-type enzyme. Our results indicate that the decreased BCAT activity enhanced BCHA production by reducing BCAA biosynthesis, which occurs via a pathway that directly competes with BCHA production. IMPORTANCE Recently, several studies have attempted to increase the production of branched-chain higher alcohols (BCHAs) in the yeast Saccharomyces cerevisiae. The key enzymes for BCHA biosynthesis in S. cerevisiae are the branched-chain amino acid aminotransaminases (BCATs) Bat1 and Bat2. Deletion or overexpression of the genes encoding BCATs has an impact on the production of BCHAs; however, amino acid substitution variants of Bat1 and Bat2 that could affect enzymatic properties-and ultimately BCHA productivity-have not been fully studied. By using in silico analysis, we designed variants of Bat1 and Bat2 and expressed them in yeast cells. We found that the engineered BCATs decreased catalytic activities and increased BCHA production. Our approach provides new insight into the functions of BCATs and will be useful in the future construction of enzymes optimized for high-level production of BCHAs.
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Project description:There is significant interest in the wine industry to develop methods to reduce the ethanol content of wine. Here the global transcription machinery engineering (gTME) technology was used to engineer a yeast strain with decreased ethanol yield, based on the mutation of the SPT15 gene. We created a strain of Saccharomyces cerevisiae (YS59-409), which possessed ethanol yield reduced by 34.9%; this was accompanied by the increase in CO2, biomass, and glycerol formation. Five mutation sites were identified in the mutated SPT15 gene of YS59-409. RNA-Seq and metabolome analysis of YS59-409 were conducted compared with control strain, suggesting that ribosome biogenesis, nucleotide metabolism, glycolysis flux, Crabtree effect, NAD+/NADH homeostasis and energy metabolism might be regulated by the mutagenesis of SPT15 gene. Furthermore, two genes related to energy metabolism, RGI1 and RGI2, were found to be associated with the weakened ethanol production capacity, although the precise mechanisms involved need to be further elucidated. This study highlighted the importance of applying gTME technology when attempting to reduce ethanol production by yeast, possibly reprogramming yeast's metabolism at the global level.
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Project description:Rice wine, well known for its unique flavor, rich nutritional value, and health benefits, has potential for extensive market development. Rhizopus and Aspergillus are among several microorganisms used in rice wine brewing and are crucial for determining rice wine quality. The strains were isolated via Rose Bengal and starch as a combined separation medium, followed by oenological property and sensory evaluation screening. The strain exhibiting the best performance can be screened using the traditional rice wine Qu. The strains YM-8, YM-10, and YM-16, which exhibited strong saccharification and fermentation performance along with good flavor and taste, were obtained from traditional rice wine Qu. Based on ITS genetic sequence analysis, the YM-8, YM-10, and YM-16 strains were identified as Rhizopus microsporus, Rhizopus arrhizus, and Aspergillus oryzae. The optimum growth temperature of each of the three strains was 30°C, 32°C, and 30°C, and the optimum initial pH was 6.0, 6.5, and 6.5, respectively. The activities of α-amylase, glucoamylase, and protease of YM-16 were highest at 220.23±1.88, 1,269.04±30.32, and 175.16±1.81 U/g, respectively. The amino acid content of rice wine fermented in a 20-L bioreactor with the three mold strains was higher than that of the control group, except for arginine, which was significantly lower than that of the control group. The total amino acid content and the total content of each type of amino acid were ranked as YM-16 > YM-8 > YM-10 > control group, and the amino acid content varied greatly among the strains. The control group had a higher content, whereas YM-8 and YM-16 had lower contents of volatile aroma components than the control group and had the basic flavor substances needed for rice wine, which is conducive to the formation of rice wine aroma. This selected strain, YM-16, has strong saccharification and fermentation ability, is a rich enzyme system, and improves the flavor of rice wine, thereby demonstrating its suitability as a production strain for brewing.
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Project description:Copper is widely used in agriculture as a traditional fungicide in organic farming to control downy mildew on grapes, consequently it is possible to find this metal during all stages of the vinification process. Low amounts of copper play a key role on the function of key cell enzymes, whereas excess quantities can exert amount-dependent cytotoxicity, resulting in general cellular damage. Nowadays the excessive copper ions in wines is removed by addition of adsorbents, but these additives can influence the sensory characteristics of wine, as well as detrimental to the health of consumers. It is well known that high concentrations of Cu2+ can be toxic to yeasts, inhibiting growth and activity, causing sluggish fermentation and reducing alcohol production. In this study, 47 S. cerevisiae strains were tested for copper tolerance by two different tests, growth on copper added medium and fermentative activity in copper added grape must. The results obtained by the two different tests were comparable and the high strain variability found was used to select four wild strains, possessing this characteristic at the highest (PP1-13 and A20) and the lowest level (MPR2-24 and A13). The selected strains were tested in synthetic and natural grape must fermentation for ability to reduce copper content in wine. The determination of copper content in wines and yeast cells revealed that at the lowest copper residual in wine corresponded the highest content in yeast cells, indicating a strong strain ability to reduce the copper content in wine. This effect was inversely correlated with strain copper resistance and the most powerful strain in copper reduction was the most sensitive strain, MPR2-24. This wild strain was finally tested as starter culture in cellar pilot scale fermentation in comparison to a commercial starter, confirming the behavior exhibited at lab scale. The use of this wild strain to complete the alcoholic fermentation and remove the copper from wine represents a biotechnological sustainable approach, as alternative to the chemical-physical methods, ensuring at the same time a completed alcoholic fermentation and organoleptic quality of wine.
| S-EPMC5767583 | biostudies-literature
Project description:The structure of yeast cell wall (CW) mannoproteins (MPs) influences their impact on wine properties. Yeast species produce a diverse range of MPs, but the link between properties and specific structural features has been ill-characterized. This study compared the protein and polysaccharide moieties of MP-rich preparations from four strains of four different enologically relevant yeast species, named Saccharomyces boulardii (SB62), Saccharomyces cerevisiae (SC01), Metschnikowia fructicola (MF77), and Torulaspora delbrueckii (TD70), and a commercial MP preparation. Monosaccharide determination revealed that SB62 MPs contained the highest mannose/glucose ratio followed by SC01, while polysaccharide size distribution analyses showed maximum molecular weights ranging from 1349 kDa for MF77 to 483 kDa for TD70. Protein identification analysis led to the identification of unique CW proteins in SB62, SC01, and TD70, as well as some proteins shared between different strains. This study reveals MP composition diversity within wine yeasts and paves the way toward their industrial exploitation.
| S-EPMC10722544 | biostudies-literature
Project description:Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density.We detected eight genomic regions explaining the diversity concerning 15 compounds, some produced de novo by yeast, such as nerolidol, ethyl esters and phenyl ethanol, and others derived from grape compounds such as citronellol, and cis-rose oxide. In three of these eight regions, we identified genes involved in the phenotype. Hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the PDR8 and ABZ1 genes, respectively. Deletion of a PLB2 gene confirmed its involvement in the production of ethyl esters. A comparison of allelic variants of PDR8 and ABZ1 in a set of available sequences revealed that both genes present a higher than expected number of non-synonymous mutations indicating possible balancing selection.This study illustrates the value of QTL analysis for the analysis of metabolic traits, and in particular the production of wine aromas. It also identifies the particular role of the PDR8 gene in the production of farnesyldiphosphate derivatives, of ABZ1 in the production of numerous compounds and of PLB2 in ethyl ester synthesis. This work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide.
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Project description:In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with Saccharomyces cerevisiae, to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species (S. cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans, Hanseniaspora uvarum, Candida sake and Metschnikowia pulcherrima) in synthetic grape must. Proteomic analysis of EV-enriched fractions from S. cerevisiae and T. delbrueckii showed enrichment in glycolytic enzymes and cell-wall-related proteins. The most abundant protein found in S. cerevisiae, T. delbrueckii and L. thermotolerans EV-enriched fractions was the enzyme exo-1,3-β-glucanase. However, this protein was not involved in the here-observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology.
| S-EPMC7415363 | biostudies-literature
Project description:ObjectiveCompared with standard wines, low-alcohol wines may have several social and health benefits. Innovative production processes have led to high-quality light wines. It is, however, unclear how consumers perceive and consume these alcohol-reduced wines. The current study aimed to investigate how people evaluate low-alcohol wine (Sauvignon Blanc) and if the reduction in alcohol and the information that a wine is low in alcohol influences consumption.DesignRandomised controlled trial (RCT).SettingParticipants were invited to a wine tasting and randomised into one of the three conditions: they either tasted a 'new white wine' (12·5 % alcohol content), a 'new low-alcohol white wine' (8·0 % alcohol content) or they tasted the low-alcohol wine but were not aware that the wine was reduced in alcohol (low-alcohol, blinded).ParticipantsNinety participants (42 % male, mean age = 41 (sd 14) years).ResultsMean comparisons showed similar ratings for the low-alcohol conditions and the standard alcohol condition (mean > 5·6/7). The mean consumed amount across all conditions did not differ (162 (sd 71) ml, (F2,86 = 0·43, P > 0·05)), hence people who tasted the low-alcohol wine consumed approximately 30 % less alcohol. However, participants were willing to pay more for the normal wine compared with the low-alcohol wine, (F2,87 = 3·14, P < 0·05).ConclusionsParticipants did not alter their drinking behaviour in response to the reduced alcohol content, and the low-alcohol wine was perceived positively. There might be an emerging market potential for wine of reduced alcohol content, but consumers may not be willing to pay the same price as for the standard wine.
| S-EPMC10200567 | biostudies-literature
Project description:Chinese rice wine (CRW) is the oldest kind of wine in China and is mainly fermented by wheat Qu and yeast with rice, millet, etc. This gives CRW a unique quality, but the flavor components are complex. Its formation is related to microorganisms, but the link between CRW and microorganisms is poorly understood. Here, we used two kinds of sorghum (JZ22 and JB3, of which JZ22 has a higher tannin content) as the raw materials to brew and determined the structural and functional dynamics of the microbiota by metagenomics and flavor analyses. We detected 106 (JZ22) and 109 (JB3) volatile flavor compounds and 8 organic acids. By correlation analysis, we established 687 (JZ22) and 496 (JB3) correlations between the major flavor compounds and microbes. In JZ22, Blautia, Collinsella, Bifidobacterium, Faecalibacterium and Prevotella had the most correlations with flavor production. In JB3, the top 5 genera were Stenotrophomonas, Bdellovibrio, Solibacillus, Sulfuritalea and Achromobacter. In addition, more esters were detected, and more microorganisms correlated with ester generation in JZ22. This study provides a new idea for the micro ecological diversity of CRW fermented with sorghum. This is of significance for improving the quality and broadening the CRW varieties.
| S-EPMC5854674 | biostudies-literature