GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non-utilizing phenotype. A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

高乙醇转化率酿酒酵母工程菌株构建研究进展

酿酒酵母(Saccharomyces cerevisiae)发酵产生乙醇的过程中,甘油的生成所消耗的碳源约占总碳源的4%~10%。减少甘油合成量可提高乙醇产率与碳源利用率。其主要策略是修饰或切除一步或多步代谢反应,或引入外源相关基因以改变碳流方向与碳流量,从而使反应向有利于生成更多乙醇而少生成甘油的方向进行。文中主要综述了近年来通过代谢工程手段阻断酿酒酵母甘油的合成或降低甘油的合成量,以提高乙醇发酵糖醇转化率的研究进展。     

桓仁产区优良冰酒酵母菌株的筛选及酿造特性评价（英文）

为获得可用于东北桓仁地区威代尔冰酒生产的酿酒酵母菌株,采用一种快速的酵母菌株筛选方法,通过测定菌株乙醇和二氧化硫耐受性,从威代尔冰酒自然发酵过程中筛选到9株酿酒酵母菌株。进一步酿造实验结果显示,所筛选的酿酒酵母可以顺利完成冰酒发酵,产生的香气轮廓与商业酵母DV10相比也不同(主成分分析结果),最终获得了2株具有高发酵活力且香气特征与商业酵母差异显著的酵母菌株SC42和SC45,其中SC42能够高产高级醇和酯类物质,并且低产乙酸,而SC45能够产生高含量的甘油、酯类物质以及反式玫瑰醚和β-大马士酮。结果表明,采用本研究的筛选方法能够快速有效地筛选到具有应用潜力的冰酒生产菌株,同时也证明了使用本土野生酵母菌株能够有效地改善冰酒香气品质,生产出与接种商业酵母不同风格的冰酒产品。     

Changes in the Lipid Composition of Saccharomyces cerevisiae Race Capensis (G-1) During Alcoholic Fermentation and Flor Film Formation

The cellular lipid composition of one flor-forming strain of Saccharomyces cerevisiae during fermentation and the subsequent period of film formation with different oxygen levels was studied. Irrespective of fermentation conditions, only those yeasts which came into contact with oxygen after fermentation formed a flor film. After the fermentation, these yeasts entered an adaptation phase in which the percentage of oleic acid increased considerably at the expense of other long-chain fatty acids. Their phospholipid contents remained high, as well as the unsaturation index of their fatty acids and the ergosterol/phospholipids ratio was maintained below 1. These changes allowed an increased viability of yeasts in the wine of up to 80% and the acquisition of sufficient hydrophobicity and floatability to reach the surface and form flor film.     

在酿酒酵母中共表达sXYLA和XKS1及木糖发酵的初步研究

为改善重组酵母发酵木糖生产乙醇的能力,将定点突变改造后的Thermus thermophilus木糖异构酶基因sXYLA克隆到酵母表达载体pYX212并用于转化酸酒酵母Saccharomyces cerevisiae YPH499进行表达研究。酶活检测表明,改造后的木糖异构酶活性是未改造的1.91倍。在此基础上将改造后具有良好特性的木糖异构酶基因sXYLA和来自酸酒酵母的木酮糖激酶基因XKS1耦联,构楚得到重组表达质粒pYX-sXYLA- XKS1,在酿酒酵母YPH499中实现组成型共表达。结果表明,在84 h时重组菌发酵液酶活达到最高,木糖异构酶为0.624 U/mg蛋白,木酮糖激酶为0.688 U/mg蛋白。以葡萄糖和木糖为混合碳源初步进行半通氧发酵,代谢产物分析表明酸酒酵母重组菌木糖的消耗为4.75 g/L,乙醇的产量为0.839 g/L,分别比出发菌提高20.9%和14.8%,为酿酒酵母利用木糖发酵乙醇奠定基础。     

共表达xyl1、xyl2和tal1重组酿酒酵母的构建及木糖发酵研究

将来自树干毕赤酵母的木糖还原酶基因(xyl1)、木糖醇脱氢酶基因(xyl2)和酿酒酵母自身的木糖转醛酶基因(tal1),通过串联共表达的方法构建到表达载体pAUR123上,构建了1株重组酿酒酵母。该菌在打通木糖向木酮糖转化通路基础上,超表达木糖转醛酶。该菌以木糖为唯一碳源进行限氧发酵,能初步利用木糖。结果表明,木糖的利用率为77.4%,但乙醇产率仅为0.04 mg/mL。同时探讨了3种酶共表达对酿酒酵母发酵木糖生成乙醇的影响,发现3种酶对木糖的利用起关键性的作用,其过量表达导致木糖醇大量积累,乙醇得率降低。     

Determination of the Relative Ploidy in Different Saccharomyces cerevisiae Strains used for Fermentation and ‘Flor’ Film Ageing of Dry Sherry-type Wines

The full chromosomal karyotype of six enological Saccharomyces cerevisiae strains used for fermentation and biological ageing of sherry-type wines was studied. A genetic method based on the analysis of segregation frequencies of auxotrophic markers, among random spore progeny of hybrids, constructed between laboratory and industrial wine strains (Bakalinsky and Snow, 1990) was used. This method was combined with the analysis of strains by pulsed-field gel electrophoresis. The results obtained clearly indicate the presence of two, three or four copies of a chromosome in the industrial strains examined, and thus confirm that aneuploidy/polyploidy is not uncommon in these strains. In all strains examined, chromosome XIII polysomy is observed. This chromosome contains the ADH2 and ADH3 loci, that code for the ADHII and ADHIII isoenzymes of alcohol dehydrogenase, which are involved in ethanol oxidative utilization during biological ageing of wines. Tetrad analysis for the ‘flor formation’ character suggests two possibilities: this character is either regulated by at least a digenic system, or by only one gene present on a chromosome which is, at least, disomic.© 1997 John Wiley & Sons, Ltd.     

Glycosylation of human α1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Human α₁-antitrypsin (α₁-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α₁-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α₁-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α₁-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α₁-AT. The human α₁-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α₁-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α₁-AT in S. cerevisiae. The α₁-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α₁-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.     

A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1

Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.     

The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol Δ⁵ desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13·7 centimorgans from GAL2 toward SPT8. The essentially of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without ‘sparking’ levels of Δ⁵ sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

The sequence of a 17 933 bp segment of Saccharomyces cerevisiae chromosome XIV contains the RHO2, TOP2, MKT1 and END3 genes and five new open reading frames

We report the DNA sequence of a 17 933 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Analysis of the sequence reveals the presence of ten open reading frames (ORFs) larger than 100 codons. Four of these were previously identified as genes RHO2, TOP2, MKT1 and END3. Additionally, the NH₂ end coding region of PMS1 is found in the 3′ end of the sequence. No significant homology to any known protein has been found for the other five ORFs. The nucleotide sequence has been deposited at EMBL, with Accession Number X89016.     

The Genes Encoding the Transcription Factor yTAFII60, the G4p1 Protein and a Putative Glucose Transporter are Contained in a 12·3 kb DNA Fragment on the Left Arm of Saccharomyces cerevisiae Chromosome VII

We report the nucleotide sequence of a DNA fragment of 12 325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAF_II60. The G3075 ORF is 47·8% identical to the hypothetical yeast protein YB88. G3080 shows 36·7% identity to the eel calmodulin. G3085 shows 94·9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46·7% identity with the probable glucose transport protein yBR1625. The DNA sequence has been submitted to the EMBL data library under Accession Number X97644. © 1997 by John Wiley & Sons, Ltd.     

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimised methodology for carboxymethylation of (1→3)‐β‐d‐glucan from Yeast (Saccharomyces cerevisiae) and promotion of mechanical activation

With a view to utilise yeast (1→3)‐β‐d ‐glucan as biological response modifiers with better water solubility, carboxymethylation was carried out by a two‐step alkalisation and etherification with monochloroacetic acid. Four technological parameters of carboxymethylation were investigated by orthogonal experiments for obtaining the maximum degree of substitution (DS), apparent viscosity (η) and solubility of carboxymethyl derivatives. In view of the orthogonal analysis, the optimal technological parameters were reaction temperature 50 °C, total reaction time 5 h, 3 mL of 50% sodium hydroxide as the second alkali dosage and 15 mL of 4 m chloroacetic acid. In addition, it was found that ball milling pretreatment for original (1→3)‐β‐d ‐glucan can be an advantage for carboxymethylation. By contrast, DS, η and solubility of carboxymethyl product increased 24%, 6% and 22%, respectively, suggesting the effect of ball milling pretreatment could not be neglected on improvement of DS, η and solubility for carboxymethyl products.