Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Delta strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.     

Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain of Saccharomyces cerevisiae

Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.Flor or velum formation by certain wine strains of Saccharomyces cerevisiae (flor strains) is a form of cellular aggregation observed as an air-liquid interfacial biofilm at the end of the alcoholic fermentation. Formation of the biofilm appears to be an adaptive mechanism because it ensures access to oxygen and therefore permits continued growth on nonfermentable ethanol. In general, nonbuoyant cells cease growth at the end of completed wine fermentations not for lack of carbon but for lack of oxygen. Biofilm cells have been found to have an elevated and/or altered lipid content and increased surface hydrophobicity (3, 5, 8, 9, 11). While both Hsp12, a small heat shock protein (13), and Muc1 (also known as Flo11), a hydrophobic cell wall mannoprotein (4, 6), have been shown to be required for the flor biofilm (10, 12, 14), other genetic or environmental requirements, other than an absence of glucose and the presence of ethanol and oxygen, have not been demonstrated. Here, we asked whether flor formation could be induced during growth on nonfermentable substrates other than ethanol. On the basis of dry weight of biofilm formed per mg of available carbon, the best carbon sources were found to be glycerol, ethyl acetate, and ethanol, in descending order. While subsurface growth occurred on acetic, dl-lactic, and succinic acids, an air-liquid interfacial biofilm did not always form. Microarray analysis of cells shifted from growth on glucose to growth on ethanol did not detect significant changes in expression of known biofilm formation-associated genes.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

Cytokinin Utilization by Adenine-Requiring Mutants of the Yeast Saccharomyces cerevisiae

By monitoring the growth of several adenine auxotrophs of the yeast Saccharomyces cerevisiae on cytokinin-supplemented media, we have demonstrated that this organism can utilize some of these derivatives as a source of adenine. Growth of a mutant lacking adenylosuccinate synthetase suggests that the conversion of cytokinins to adenine does not involve a hypoxanthine intermediate and may be catalyzed by an enzyme analogous to cytokinin oxidase.     

Engineering a Saccharomyces cerevisiae Wine Yeast That Exhibits Reduced Ethanol Production during Fermentation under Controlled Microoxygenation Conditions

We recently showed that expressing an H₂O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD⁺ reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

Genetic Determinants of Volatile-Thiol Release by Saccharomyces cerevisiae during Wine Fermentation

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.     

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO₂ production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.     

赤霞珠葡萄酒自然发酵中酿酒酵母的菌株区分

采用Interdelta指纹图谱分析, 对分离自宁夏地区赤霞珠葡萄自然发酵过程中的45个酿酒酵母单菌落进行菌株区分, 研究发酵过程中酿酒酵母菌株的变化, 为发酵的有效控制及选育优良酿酒酵母菌株提供依据。结果发现, 本研究分离到的45个酿酒酵母单菌落中, 产生5种指纹图谱, 代表5种不同的基因型, 基因型I-V分别占所分离单菌落的71%、13%、9%、5.0%、2.0%, 基因型I是发酵过程中的优势菌株。本研究中, 二氧化硫处理影响自然发酵过程中酿酒酵母菌株的类型、数目及比例, 但其影响不是很大。     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K_m and 4-fold higher V_max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains.     

Improvement of a Wine Saccharomyces cerevisiae Strain by a Breeding Program

Hybridization by spore conjugation was used to develop new and improved wine yeasts of Saccharomyces cerevisiae. The procedure was achieved with diploid, homothallic strains with high sporulation frequency and high spore viability. The method was verified by crossing flocculent and non-H₂S-forming strains. Single-spore descendants of the hybrids were studied by tetrad analysis with regard to the aforementioned characters and the other two winemaking traits, i.e., ethanol production and fermentation rate. A highly flocculent, non-H₂S-forming wine yeast strain with a high fermentation rate and high ethanol production was obtained.     

Peculiar H+ Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation

Intracellular pH (pH(in)) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H(+) homeostasis is accomplished by an industrial wine strain of Saccharomyces cerevisiae while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH(in) 5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H(+) via H(+)-ATPase and of keeping H(+) out by means of an impermeable membrane were evaluated as potential mechanisms of H(+) homeostasis. At 30°C, H(+) efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H(+). Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H(+) remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H(+) permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H(+) homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.     

Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.     

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.     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.     

Utilization of D-asparagine by Saccharomyces cerevisiae.

Yeast strains sigma1278b and Harden and Young, which synthesize only an internal constitutive form of L-asparaginase, do not grow on D-asparagine, as a sole source of nitrogen, and whole cell suspensions of these strains do not hydrolyze D-asparagine. Strains X2180-A2 and D273-10B, which possess an externally active form of asparaginase, are able to grow slowly on D-asparagine, and nitrogen-starved suspensions of these strains exhibit high activity toward the D-isomer. Nitrogen starvation of strain X218O-A2 results in coordinate increase of D- and L-asparaginase activity; the specific activity observed for the D-isomer is approximately 20% greater than that observed for the L-isomer. It was observed, in studies with cell extracts, that hydrolysis of D-asparagine occurred only with extracts from nitrogen-starved cells of strains that synthesize the external form of asparaginase. Furthermore, the activity of the extracts toward the D-isomer was always higher than that observed with the L-isomer. A 400-fold purified preparation of external asparaginase from Saccharomyces cerevisiae X218U-A2 hydrolyzed D-asparagine with an apparent Km of 0.23 mM and a Vmax of 38.7 mumol/min per mg of protein. D-Asparagine was a competitive inhibitor of L-asparagine hydrolysis and the Ki determined for this inhibition was approximately equal to its Km. These data suggest that D-asparagine is a good substrate for the external yeast asparaginase but is a poor substrate for the internal enzyme.