The Production of Volatile Compounds by Yeasts Isolated from Small Brazilian cachaça distilleries

Summary The production of volatile compounds by 24 strains of Saccharomyces cerevisiae and one strain each of Candida apicola, C. famata, C. guilliermondii, Hanseniaspora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe was evaluated with respect to the production of cacha?a. They were isolated from small cacha?a distilleries (27), industrial cacha?a distilleries (2) and one sugarcane alcohol distillery, and tested in synthetic medium for the production of acetaldehyde, ethyl acetate, propanol, isobutanol, isoamyl alcohol, acetic acid and glycerol. The Saccharomyces strains showed a narrow range of variation in the production of such compounds, near 50% of the average of each volatile compound concentration. Principal component analysis showed the separation of the strains into six groups, and acetic acid production was the variable of greatest impact in the differentiation of the strains. The strains of S. pombe formed a distinct group (Group 2), and the strains of C. apicola and H. occidentalis formed a joint group (Group 6) as did Sc13 and Sc4 (Group 4). Group 1 was formed exclusively of S. cerevisiae. The closest non-Saccharomyces strains were C. apicola and H. occidentalis, with a similarity index of about 0.95. The strain P. subpelliculosa showed general characteristics more similar to those of the S. cerevisiae strains than to the non-Saccharomyces strains.     

Immobilization of Saccharomyces Cerevisiae Cells by Gel Entrapment Using Various Metal Alginates

Summary Immobilization of whole yeast cells (Saccharomyces cerevisiae) in calcium alginate is well known. The present work describes the feasibility of gel entrapment of whole cell yeast using strontium, barium, calcium–strontium, calcium–barium and strontium–barium alginates.     

Development of novel carrier using natural zeolite and continuous ethanol fermentation with immobilized Saccharomyces cerevisiae in a bioreactor

A natural zeolite, easily vitrified and blown at 1300 °C with a high porosity and diam. of 5–100 m, was used to immobilize Saccharomyces cerevisiae at 3.6 × 10⁸ cells ml^–1 carrier. When the abilities of natural zeolite carrier were compared with glass beads, the capacity for immobilization and alcohol fermentation activity were, respectively, 2-fold higher and 1.2-fold higher than that of glass beads. Continuous alcohol fermentation was stable for over 21 d without breakage of the carrier.     

酵母表面展示系统研究进展

酵母表面展示系统是继噬菌体展示技术创立后发展起来的真核展示系统,酵母的蛋白质折叠和分泌机制与哺乳动物细胞非常相似,对人的蛋白质表达和展示更具优越性.酵母细胞颗粒大,可用流式细胞仪进行筛选和分离.目前报道的两种酵母展示系统分别以α或a凝集素作为融合骨架.在蛋白质的定向进化、口服疫苗的研制等多方面均有报道.     

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function

Predicted protein sequences of fungal chitin synthases can be divided into a non-homologous N-terminal region and a C-terminal region that shows significant homology among the various synthases. We have explored the function of these domains by constructing a series of nested deletions, extending from either end, in theCHS1 andCHS2 genes ofSaccharomyces cerevisiae. In both cases, most or all of the sequences encoding the non-homologous N-terminal region (one-third of the protein for Chs1p and about one-fourth for Chs2p) could be excised, with little effect on the enzymatic activity in vitro of the corresponding synthase or on its function in vivo. However, further small deletions (20–25 amino acids) into the homologous region were deleterious to enzymatic activity and function, and often led to changes in the zymogenic character of the enzymes. Similarly, relatively small (about 75 amino acids) deletions from the C-terminus resulted in loss of enzymatic activity and function of both synthases. Thus, it appears that all the information necessary for membrane localization, enzymatic activity and function resides in the homologous regions of Chs1p and Chs2p, a situation that may also apply to other chitin synthases.These authors contributed equally to this paper     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。     

酿酒酵母中SSK2基因与其他镉耐受相关基因之间的双基因缺失菌株构建

镉是一种严重的环境污染物,对人体具有致癌性,能蓄积在生物体内影响机体的生长、发育和生殖。有丝分裂原蛋白激酶(Mitogen-activated protein kinase,MAPK)在调节细胞存活、增殖和分化中是重要的信号分子,并能够被镉胁迫激活。酿酒酵母中2个MAPK信号传导途径,高渗透压甘油(High Osmolarity Glycerol,HOG)途径和细胞壁完整性(Cell Wall Integrity,CWI)途径都参与Cd2+胁迫下的细胞应答。为了进一步研究这两条途径在调控Cd2+胁迫方面的相互作用,以HOG途径的蛋白激酶SSK2基因为例,通过合成遗传阵列(Synthetic Genetic Array,SGA)方法,成功构建了SSK2基因与其他52个Cd2+耐受相关基因之间的双基因缺失菌株。为大规模研究Cd2+耐受基因之间在调控镉胁迫方面的遗传学相互作用奠定了基础,也为酿酒酵母的相关研究提供了一个新的遗传学手段。     

The Capacity of Manno-Oligosaccharides,Thermolysed Yeast and Active Yeast to Attenuate Aflatoxicosis

This study assessed the ability of thermolysed, active yeast, Saccharomyces cerevisiae, and manno-oligosaccharides, to reduce the effects of aflatoxins in animals. A basic ration was developed with six different formulations. Each formulation was considered a treatment. The treatments were: an aflatoxin-free formulation, an aflatoxin control (400 g kg^–1) and four aflatoxin-supplemented formulations. The supplements were 0.1 and 0.2% manno-oligosaccharides, 1% thermolysed yeast and 1% dehydrated active yeast. The experiment was randomly designed and had five repetitions per treatment. The feed was contaminated with aflatoxins from naturally contaminated peanuts. A bioassay with Wistar rats was conducted after 28 days. The aflatoxin toxicity was evaluated by weighing body organs (heart, kidneys and liver) and by analysing the liver tissue of the animals. No significant differences were observed for the weights of the body organs from the animals fed with the different rations. However, the analysis of the liver tissue showed animals fed with 400 g of aflatoxin kg^–1, and those fed diets with aflatoxin amended with either manno-oligosaccharides or with thermolysed yeast had clear signs of toxicity and damage, while those fed with dehydrated active yeast showed less intense toxicity and less liver damage. Therefore, the thermolysed yeast and manno-oligosaccharides did not suppress damage to liver tissue caused by aflatoxins, while active yeast reduced the aflatoxin symptoms in the hepatocytes.     

酵母PMT1与PMT5双基因缺失对细胞复制性寿命的影响

为研究蛋白质O-甘露糖转移酶-1(Protein O-mannosyltransferase-1,Pmt1p)与Pmt5p基因对酵母细胞寿命的影响,采用一步基因置换法构建PMT5基因缺失菌株(pmt5Δ),在此基础上,缺失PMT1基因,构建PMT1和PMT5双基因缺失菌株(pmt1Δpmt5Δ)。显微镜下分离和计数酵母子细胞的数目,统计菌株的复制性寿命;检测细胞吸光度值来评价细胞分裂增殖速度。结果发现,与对照组酵母细胞的平均复制性寿命(25代)比较,pmt5Δ菌株(26代)的寿命无明显变化(P 0. 05),而pmt1Δpmt5Δ菌株(21代)的寿命明显缩短(P 0. 01);热量限制条件下,与对照组酵母细胞比较,pmt5Δ菌株的生长曲线无明显变化,而pmt1Δpmt5Δ菌株的生长曲线低平,细胞分裂增殖减慢。结果表明,PMT1与PMT5双基因缺失明显缩短酵母细胞的寿命,机制可能与细胞的增殖活力下降有关。     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Yeast cytochrome c is a sequence-specific DNA-binding protein

A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

The Role of Amino-Terminal and Carboxy-Terminal Extensions in the Processing and Translocation of a Plant Proteinase (Actinidin) Expressed in the Yeast Saccharomyces cerevisiae

Summary A plant proteinase gene naturally occuring in the Kiwi fruit plant (Actinidia chinensis) has been expressed in a yeast Saccharomyces cerevisiae. Different gene constructions consisting of different portions of the whole actinidin-encoding gene have been created and expressed using an expression-secretion yeast vector. It was observed that the amino- and carboxy-terminal extensions of the actinidin-encoding gene were required for the correct expression of the gene in yeast. A gene construction lacking both amino- and C-terminal extensions did not result in a detectable protein product. Similarly, a gene construction consisting of the amino-terminal extension plus mature actinidin-encoding DNA did not result in a detectable expression. However, intracellular expression was observed when a gene construction consisting of mature actinidin-encoding DNA plus C-terminal extension portion was employed. The expressed polypeptide was found however not to be correctly processed as it had a bigger size than the native actinidin. The correctly processed polypeptide was expressed intracellularly when the full-length actinidin cDNA was expressed in a vacuolar protease-proficient yeast strain. However, when a vacuolar protease-deficient yeast strain was employed, it was found that the precursor protein was not correctly processed, suggesting that the actinidin precursor had entered the vacuole and undergone proteolytic processing. The full-length actinidin cDNA consisted of the amino-terminal extension DNA, mature actinidin-encoding DNA, and C-terminal extension DNA. The results thus suggested that both amino- and C-terminal extensions were required for correct expression and processing of actinidin in yeast. The intracellular expression also suggested that the actinidin-encoding sequences contain intracellular targeting sequences which override the secretion signal included in the expression-secretion vector.     

走出青藏高原：拉格啤酒酵母的起源与演化

采用低温底层发酵的拉格(lager)啤酒15世纪开始在德国巴伐利亚地区出现,19世纪初流行至全世界,目前已成为全球产量最高的酒精饮料。目前已阐明,拉格啤酒发酵酵母为巴斯德酿酒酵母(Saccharomyces pastorianus),该种是一个杂交种,由艾尔(ale)啤酒酵母(Saccharomyces cerevisiae)与野生真贝氏酿酒酵母(Saccharomyces eubayanus)杂交而成,后者赋予了拉格啤酒酵母的耐低温能力。近年的群体遗传学和群体基因组学研究表明,拉格啤酒酵母的野生亲本S.eubayanus起源于青藏高原,可能通过丝绸之路传播到了欧洲。比较基因组学研究表明,拉格啤酒酵母包含2个株系,即Ⅰ系/Saaz系和Ⅱ系/Frohberg系,早期分别流行于中欧和西欧地区。前者为近似异源3倍体,后者为近似异源4倍体。2个株系在耐低温、麦芽三糖利用和风味物质产生能力等方面具有明显差异。在中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center,CGMCC)保藏的S.pastorianus...     

酱香型白酒堆积酒醅中象牙色克罗彭斯特德菌的分离筛选及其代谢特性解析

【目的】高温堆积发酵是酱香型白酒生产中的关键工艺环节,克罗彭斯特德菌属(Kroppenstedtia)是堆积酒醅中的优势细菌属,研究其生长和代谢特征对于理解堆积发酵的关键作用至关重要。【方法】采用胰酪大豆蛋白胨(tryptic soy broth, TSB)培养基从酒醅中筛选克罗彭斯特德菌,通过形态学和16S rRNA基因测序确定其分类学地位,结合菌株纯培养及不同温度(45 ℃和50 ℃)下的高粱固态发酵实验,研究其生长和挥发性化合物代谢特征。【结果】从酱香型白酒堆积酒醅中分离筛选到3株克罗彭斯特德菌,经鉴定为象牙色克罗彭斯特德菌(Kroppenstedtiaeburnea)。液态培养时菌株K. eburnea 1613促进吡嗪类物质的产生,为对照的2.66倍。在固态发酵高粱中检出的挥发性化合物主要为醇类和酸类,总含量随发酵时间的推移而逐渐增加。50 ℃发酵高粱有利于醇类、酸类和吡嗪类物质的积累,而45 ℃下酯类物质含量较高。3株菌以高粱为基质进行固态发酵时主要代谢产物是苯乙醇和异戊酸,其中K. eburnea 1615在50 ℃下发酵15 d时苯乙醇和异戊酸含量最高,为(31.17±0.14) µg/g和(16.75±0.76)µg/g。菌株K. eburnea 6E22在50℃下发酵15 d时2,5-二甲基吡嗪含量最高,为(1.67±0.14) µg/g。菌株K. eburnea 1613在发酵15 d时己酸含量最高,为(3.74±0.19) µg/g。50 ℃下发酵高粱自身中醛酮类物质积累明显。偏最小二乘判别分析(partial least squares discrimination analysis, PLS-DA)显示,温度和时间对3株菌发酵高粱中挥发性化合物的组成有显著影响。【结论】象牙色克罗彭斯特德菌有助于堆积发酵酒醅风味化合物的产生,特别是醇类、酸类和吡嗪类等酱香型白酒特征风味物质。     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.