Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

利用26S rDNA D1/D2区序列和微卫星标记分析各种工业酿酒酵母的种内遗传差异

选取30株传统发酵工业中不同来源的酿酒酵母菌株和实验室常用酿酒酵母菌株,利用大亚基(26S)rDNA D1/D2区序列和微卫星标记分析酿酒酵母种内菌株间的遗传多样性和系统发育关系,以揭示酿酒酵母在长期的各种工业发酵环境下发生的遗传变异。结果表明:30株酿酒酵母的26S rDNA D1/D2区序列(591bp)比较保守,与测序菌株S288C相比序列相似度在99.8%–100%,说明种内菌株间存在一定的多态性,但序列差异并不十分明显,其变异情况表现在个别碱基的差异(大多由转换突变引起);通过扩增11个微卫星位点,每个菌株均有其独特的基因型,即30种基因型,共得到188个等位基因,观测杂合度平均值和期望杂合度平均值分别为0.576、0.886,多态信息含量平均值高达0.858,说明酿酒酵母种内菌株间具有较高的遗传多样性,而聚类分析表明30株酿酒酵母可以得到很好地区分,但是没有呈现与其工业来源相关的聚类。     

Generation of a large set of genetically tractable haploid and diploid Saccharomyces strains

Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics studies. Despite several hundreds of isolates already available, the scientific community relies on the use of only a handful of unrelated strains. The lack of sequence information, haploid derivatives and genetic markers has prevented novel strains from being used. Here, we release a set of 55 S. cerevisiae and Saccharomyces paradoxus genetically tractable strains, previously sequenced in the Saccharomyces Genome Resequencing Project. These strains are stable haploid derivatives and ura3 auxotrophs tagged with a 6-bp barcode, recognized by a restriction enzyme to allow easy identification. We show that the specific barcode can be used to accurately measure the prevalence of different strains during competition experiments. These strains are now amenable to a wide variety of genetic experiments and can be easily crossed with each other to create hybrids and segregants, providing a valuable resource for breeding programmes and quantitative genetic studies. Three versions of each strain (haploid Mat a and Mat α and diploid Mat a /α all as ura3 ∷ KanMX-Barcode ) are available through the National Culture Yeast Collection.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

基因组改组技术选育耐高温、耐高乙醇酿酒酵母菌株的研究

当以f4、f5、f6作为出发菌株,用酵母菌原生质体紫外诱变的方法,在不同温度下,用含有不同浓度乙醇的平板筛选,分别获得了在耐高温和耐乙醇性状有较大提高的f4.2、f5.1、f6.2、f4.5等正突变菌株。以这些菌株作为出发菌株,进一步用硫酸二乙酯诱变,获得了f5.1.1、f4.2.1两个乙醇耐受性能较高的菌株。在建立了上述不同突变株后,通过基因组改组(genome shuffling)的方法,将上述不同特性的菌株经过两轮genome shuffling,获得了耐高温性能和耐乙醇性能都较好的酵母菌株。经过摇瓶发酵后证明,R24株在35℃发酵过程中,发酵液中的最高乙醇浓度12.93%(W/V),比原始出发菌株f4在35℃的发酵液中最高乙醇浓度8.11%提高了近5%。     

酿酒酵母Saccharomyces cerevisiae重组菌株木糖醇发酵的初步研究

木糖还原酶催化木糖为木糖醇的反应,是木糖代谢的第一步。将木糖还原酶的原因ＸＹＬ１引入酿酒酵母中,构建得到儿表达ＸＹＬ１基因的重组酿酒酵母菌株ＨＹＥＸ２,该重组菌株的木糖还原酶比活力为７．４７Ｕ／ｍｇ。研究表明,该菌株获得转化木糖产生木糖醇的能力,当辅助碳源葡萄糖的浓度为２％,并在发酵３０ｈ左右添加木糖,木糖醇的转化率可达到０．９７ｇ／ｇ。     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

酿酒酵母中对抗真菌药物克霉唑敏感的突变菌株的筛选

克霉唑是一种临床上用于抗真菌感染的抗真菌药物,同时也具有治疗其他疾病如疟疾、脚气病、足癣和癌症的前景.通过96孔板液体培养及倍比稀释的方法,对模式真菌酿酒酵母双倍体单基因缺失株文库中的4 532个非必需基因缺失株进行初步筛选及表型验证,对筛选出的敏感菌株测定了加克霉唑和不加克霉唑细胞内的ROS水平变化.筛选得到了127...     

The selection of yeast strains for the production of premium quality South African brandy base products

One hundred seven yeast strains were screened for their ability to produce a brandy base wine of exceptional sensory quality. Volatile acids, esters and higher alcohols were quantified and the results were interpreted using a multivariate analysis of variance (MANOVA) and an average linkage cluster analysis. Significant differences between yeast strains for higher alcohol, fatty acid ester and acetate concentrations were observed. On the basis of their chemical profiles, 16 strains were selected and re-evaluated in larger-scale fermentations and subsequent double distillations. Results show that the yeast lees can have an important effect on the final concentration of higher alcohols and esters in the distillate. Highly elevated levels of ethyl acetate and iso-amyl acetate were found to be undesirable. Elevated levels of all the esters present contributed positively to the overall potential quality of the brandy base product. Too low higher alcohol concentrations were also not desirable. Sensory evaluations showed that, since the panel was composed of representatives of the three largest brandy-producing companies, each company preferred a different yeast strain most suitable for their style of brandy. For these reasons, three strains, B7, LL2 and 20-2, warranted further evaluation on a semi-commercial scale for each of the respective companies. Journal of Industrial Microbiology & Biotechnology (2000) 24, 431–440. Received 17 November 1999/ Accepted in revised form 07 March 2000     

诱变选育脂肪酶高产菌株及其酶学性质

面包干酵母(Saccharomyces cerevisiae)为出发菌株,对其进行紫外和微波复合诱变,得高产突变菌株DX213,高产突变菌株酶活力为635 U/mL,为出发菌株的1.69倍。菌株富集培养5代,遗传性状稳定。DX213菌株的最优产脂肪酶条件为:培养温度30℃和培养液pH 7.5。酶学性质研究表明:脂肪酶的最适温度40℃、最适pH为7.5、脂肪酶在40℃以下稳定。Fe3+离子对脂肪酶有激活效应,当Fe3+离子浓度为0.03 g/mL时,脂肪酶酶活力高达720 U/mL。     

Winemaking of musts at high osmotic strength by thermotolerant yeasts

Fermentation performance of 15 thermotolerant Saccharomyces cerevisiae in three musts from dried grapes at 25, 30, and 40° brix was studied. When the osmotic strength was increased, the volatile acidity and the SO₂ production in the wines also increased: in the must with 40° brix, yeasts produce from 1.63 to 3.65 g acetic acid l^–1 and from 40 to 73.6 mg SO₂ l^–1 due to osmotic stress. From 9.75 to 13.40 ethanol v/v production is observed in must at 30°brix, whereas at 40°brix there is a clear detrimental effect.     

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。     

两株高糖分利用能力的酿酒酵母呼吸突变体选育

以酿酒酵母乙醇发酵高产工业菌株MF1002为始发菌株,对其营养细胞进紫外线诱变,筛选得到两株性状稳定的呼吸缺陷型突变体MF15c和MF11a。菌体细胞对2,3,5-氯化三苯四氮唑(TTC)显色测定呼吸强度的结果表明,两突变菌株的相对呼吸强度分别只有始发菌株的57.77%和47.25%。与现有报道的呼吸缺陷突变体不同,两株突变体的细胞生长速率只在培养初期略低于始发菌株,总体生长速率与始发菌株几乎没有差异,在YPD平板上培养也不形成小菌落。比较蔗糖发酵试验表明,两株突变体的乙醇产量较始发菌株只分别略提高6.48%-6.59%(MF15c)和1.66%-1.97%(MF11a),但发酵终止的残总糖含量却显著低于始发菌株,分别减少34.85%和19.70%,发酵效率较始发菌株分别显著提高6.69%和4.71%,表明这两株突变体为新型的呼吸缺陷型突变。鉴于提高乙醇发酵的发酵效率可显著降低生产成本,认为这两株突变菌株具有较高的潜在工业利用价值。     

基于启动子和宿主改造的酿酒酵母表达系统优化研究

生物医药领域中一套高效表达系统对于重组蛋白的生产至关重要。酿酒酵母作为一种食品级真核微生物,具有繁殖迅速、培养简单、遗传操作便捷等特点,是生产重组蛋白较理想的表达系统之一。对实验室已有的p HR酿酒酵母表达系统进行优化。分别通过易错PCR技术和菌株诱变技术对酿酒酵母启动子PTEF和宿主酿酒酵母Y16进行突变改造,经筛选、鉴定获得表达性能提高的启动子PTEFV1和酿酒酵母Y16-E14、Y16-E19。随后,利用启动子PTEFV1构建以Y16-E14为宿主的p HR-N酿酒酵母表达系统,以绿色荧光蛋白和人血清白蛋白为对象,比较表达系统改造前后性能变化。结果显示p HR-N酿酒酵母表达系统无论胞内表达绿色荧光蛋白还是分泌表达人血清白蛋白的能力均较改造前明显提高。p HR-N系统为获得更多具有重要应用价值的重组蛋白提供了有利的工具。     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。