Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Assimilable nitrogen utilisation and production of volatile and non-volatile compounds in chemically defined medium by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeasts

Surveys conducted worldwide have shown that a significant proportion of grape musts are suboptimal for yeast nutrients, especially assimilable nitrogen. Nitrogen deficiencies are linked to slow and stuck fermentations and sulphidic off-flavour formation. Nitrogen supplementation of grape musts has become common practice; however, almost no information is available on the effects of nitrogen supplementation on wine flavour. In this study, the effect of ammonium supplementation of a synthetic medium over a wide range of nitrogen values on the production of volatile and non-volatile compounds by two high-nitrogen-demand wine fermentation strains of Saccharomyces cerevisiae was determined. To facilitate this investigation, a simplified chemically defined medium that resembles the nutrient composition of grape juice was used. Analysis of variance revealed that ammonium supplementation had significant effects on the concentration of residual sugar, L-malic acid, acetic acid and glycerol but not the ethanol concentration. While choice of yeast strain significantly affected half of the aroma compounds measured, nitrogen concentrations affected 23 compounds, including medium-chain alcohols and fatty acids and their esters. Principal component analysis showed that branched-chain fatty acids and their esters were associated with low nitrogen concentrations, whereas medium-chain fatty esters and acetic acid were associated with high nitrogen concentrations.     

Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.     

Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.     

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

The timing of diammonium phosphate supplementation of wine must affects subsequent H2S release during fermentation

Aims:  The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H₂S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast. Methods and Results:  Three contrasting Saccharomyces cerevisiae wine strains were used to ferment synthetic grape juice (GJ) containing 67 mg l^?1 of initial yeast assimilable nitrogen (YAN), supplied either as DAP or as mixture of amino acids. Sufficient DAP was added either prior to or 72 h after the initiation of fermentation to achieve a final YAN concentration of 267 mg l^?1. Supplementation prior to fermentation stimulated H₂S production. The results obtained in model solutions were validated using natural GJ. Conclusion:  The timing of DAP supplementation is critical for ensuring that fermentation proceeds without excessive release of H₂S. Significance and Impact of the Study:  This result has important implications for the wine‐making industry, because it highlights the value of determining the initial nitrogen level of a GJ. It raises awareness of the dependence of wine quality on the correct timing of DAP supplementation.     

Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO₂ production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.     

硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响

【目的】利用转录组测序研究硫酸锌添加提高絮凝酿酒酵母SPSC01乙酸胁迫耐性的分子机理。【方法】在10.0 g/L乙酸胁迫条件下,添加0.03 g/L硫酸锌,取对数期酿酒酵母细胞,与不添加硫酸锌的对照组细胞进行比较转录组分析。【结果】添加硫酸锌的实验组与对照组相比较,50个基因转录水平上调,162个基因转录水平下调,这些转录水平变化明显的基因涉及糖代谢、甲硫氨酸合成、维生素合成等多条代谢途径,此外,转录水平变化的基因还包括抗氧化酶基因等关键胁迫响应基因。【结论】硫酸锌添加可改变酿酒酵母全局基因转录水平,提高抗氧化酶及其他胁迫耐性相关基因的表达,影响细胞氧化还原平衡和能量代谢,通过对多基因转录的调控提高酿酒酵母乙酸耐受性。     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

The role of GAP1 gene in the nitrogen metabolism of Saccharomyces cerevisiae during wine fermentation

Aim:  The aim of this study was to analyse the relevance of the general amino acid permease gene ( GAP1 ) of the wine yeast Saccharomyces cerevisiae on nitrogen metabolism and fermentation performance.
Methods and Results:  We constructed a gap1 mutant in a wine strain. We compared fermentation rate, biomass production and nitrogen consumption between the gap1 mutant and its parental strain during fermentations with different nitrogen concentrations. The fermentation capacity of the gap1 mutant strain was impaired in the nitrogen-limited and -excessive conditions. The nitrogen consumption rate between the wild strain and the mutant was different for some amino acids, especially those affected by nitrogen catabolite repression (NCR). The deletion of GAP1 gene also modified the gene expression of other permeases.
Conclusions:  The Gap1 permease seems to be important during wine fermentations with low and high nitrogen content, not only because of its amino acid transporter role but also because of its function as an amino acid sensor.
Significance and Impact of the Study:  A possible biotechnological advantage of a gap1 mutant is its scarce consumption of arginine, whose metabolism has been related to the production of the carcinogenic ethyl carbamate.     

The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds

Nitrogen starvation may lead to stuck and sluggish fermentations. These undesirable situations result in wines with high residual sugar, longer vinification times, and risks of microbial contamination. The typical oenological method to prevent these problems is the early addition of ammonium salts to the grape juice, although excessive levels of these compounds may lead to negative consequences for the final product. This addition reduces the overall fermentation time, regardless of the time of addition, but the effect is more significant when nitrogen is added during the yeast exponential phase. In this work we analysed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion in order to understand yeast metabolic changes that lead to the adaptation to the new conditions. These studies were carried out in a synthetic must that mimics the composition of the natural must. Furthermore, we studied how this addition affects fermentative behaviour, the levels of several yeast volatile compounds in the final product, arginase activity, and the expression of several genes involved in stress response and nitrogen metabolism during vinification. We found that the nature of the nitrogen source added during yeast late exponential growth phase introduces changes to the volatile compounds profile and to the gene expression. On the other hand, arginase activity and the expression of the stress response gene ACA1 are useful to monitor nitrogen depletion/addition during growth of the wine yeast considered under our vinification conditions.     

酵母菌在红葡萄酒酒精发酵串罐中稳定性研究

在生产条件下,对酵母菌在红葡萄酒酒精发酵串罐过程中的稳定性进行了研究。结果表明,在近1个月的时间内（相当于酵母菌细胞无性繁殖了200代）,串罐过程中的酵母菌细胞不仅能保持初始酵母菌的发酵活性和优良特性的稳定性,而且由于葡萄汁的选择作用,串罐用的酵母菌细胞的发酵活性比初始酵母菌的活性更强,因而其酒精发酵的启动和速度都更快。     

Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.