Hexokinase PII: structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae.

Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling. To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis. Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues. Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein. In most of them, the catalytic activity correlated directly with their functionality in glucose signalling. However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling. This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling.     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

Human pancreatic glucokinase (GlkB) complements the glucose signalling defect of Saccharomyces cerevisiae hxk2 mutants

Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae. The recombinant protein showed similar enzyme kinetics to those described for the original enzyme. When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant. It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart.     

Inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae.

The enzymatic steps involved in the inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae have been investigated. Yeast, incubated with 2-deoxygalactose, accumulates up to 8 mM-2-deoxygalactose, 30 mM-2-deoxygalactose-1-phosphate and 0.25 mM-UDP-2-deoxygalactose and UDP-2-deoxyglucose. An inverse correlation between 2-deoxygalactose-1-phosphate content and rate of glycolysis has been observed. The intracellular concentration of glycolytic intermediates and related metabolites point to the hexokinase and phosphofructokinase steps as the targets for the inhibition of glycolysis by 2-deoxygalactose and rule out all other mechanisms that have been proposed to explain this inhibition.     

In vitro characterization of the Mig1 repressor from Saccharomyces cerevisiae reveals evidence for monomeric and higher molecular weight forms

The Mig1 DNA-binding protein of Saccharomyces cerevisiae was expressed and purified from yeast and the physical properties were characterized by several methods, including gel filtration, sucrose gradient sedimentation and native gel electrophoresis. Purified Mig1 exists as a monomer with a Stokes' radius of 48 A and a sedimentation coefficient of 3.55 S. Mig1 has an elongated shape with a frictional coefficient of 1.83. The K(d) of purified Mig1 for the SUC2 A site is 2.8 nM and for SUC2 B site 25.8 nM; these values were similar for Mig1 purified from repressed and derepressed cells. Full-length Mig1 expressed in yeast binds more tightly to SUC2 B than bacterially expressed GST-Mig1. Sucrose gradient sedimentation resolved a larger molecular weight form of Mig1 in whole-cell extracts that was not seen in purified samples and may represent a complex with another protein. This complex is found within the nucleus and is seen only in repressed cells. Mig1 exists in multiple phosphorylation states and only less phosphorylated forms of Mig1 are associated with this complex.     

The Fate of Glucose in Strains S288C and S173-6B of the Yeast Saccharomyces cerevisiae

Intracellular metabolic flux has been investigated in two strains of Saccharomyces cerevisiae grown into stationary phase under both glucose-repressed and glucose-derepressed conditions. By employing a variety of simple methodologies (manometry, enzymatic analysis and colorimetric analysis) we have been able to identify and quantitate carbon flow from glucose without the need for isotopically labelled substrate. We can account for 88–98% (depending on strain and growth conditions) of the carbon products of glucose metabolism under both glycolytic and oxidative conditions as ethanol (27–40%), carbon dioxide (15–26%), acetate (2–3%), glycerol (5–11%), glycogen (5–13%) and trehalose (9–39%).©1997 John Wiley & Sons, Ltd.     

Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae

Three regulatory genes, known to be required for glucose repression/derepression of some genes in Saccharomyces cerevisiae, were disrupted to study their effects on the carbon-source regulation of the STA2 glucoamylase gene expression. Using a STA2-lacZ fusion it was found that: (1) the MIG1 gene is dispensable for the repression of the STA2 gene; (2) there are two components in the carbon-source repression of STA2: HXK2-dependent and HXK2-independent; and (3) the HAP2 gene seems to be involved in repression rather than activation of the STA2 expression.     

Molecular analysis of the SNF8 gene of Saccharomyces cerevisiae

Mutations in the SNF8 gene impair derepresson of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26 936-dalton product. Disruption of the chromosomal locus caused a five-fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related. The complete nucleotide sequence of SNF8 has been deposited in GenBank under accession number U10361.     

酿酒酵母细胞固定化研究

初步研究了海藻酸钙固定化技术在酿酒酵母上的应用。分析在不同条件下固定化酵母的发酵性能、机械强度，并对固定化酵母与游离酵母的发酵力进行了分析。结果表明，酵母细胞固定化后，其使用寿命、发酵周期均有大幅度提高。海藻酸钠的浓度3％，氯化钙浓度为2．0mol／L，固定化温度20℃，酵母细胞的固定化效果最为理想。     

外源乙醇对果糖与葡萄糖酒精发酵的影响

研究了不同体积分数外源乙醇对酵母GJ2008利用等量果糖与葡萄糖混合发酵过程的影响,旨在为高浓度甘蔗汁酒精发酵提供参考和依据。用YPDF培养基模拟甘蔗汁,并调节乙醇体积分数为0%、6%、9%和12%,以初始酵母数为1.12×108个/mL进行酒精发酵,高效液相色谱法测定发酵过程中果糖与葡萄糖含量,并采用曲线下面积法对果糖与葡萄糖代谢曲线进行分析。在外源乙醇体积分数为0%~9%时,酵母GJ2008可以快速利用果糖和葡萄糖进行酒精发酵,但对葡萄糖有着明显的偏好;在外源乙醇体积分数为12%时,果糖和葡萄糖消耗缓慢,生物量得不到有效增加。等质量浓度果糖与葡萄糖混合发酵表明:外源乙醇对果糖代谢的影响明显大于对葡萄糖代谢的影响,外源乙醇体积分数达6%以上时,糖转化乙醇的产量减少了19%~27%。     

外部因素对酒精酵母GJ2008果糖与葡萄糖酒精发酵的影响

在高体积分数乙醇胁迫下,考察糖比例、微通氧和温度等外部因素对果糖与葡萄糖利用及其差异性的影响,旨在为高浓度甘蔗汁发酵生产酒精提供参考和依据。用YPDF培养基模拟甘蔗汁,调节乙醇体积分数为11.2%,初始酵母数为1.40×108个/m L进行酒精发酵,测定发酵过程中果糖与葡萄糖质量分数,并采用曲线下面积法对果糖与葡萄糖代谢曲线进行分析。在高体积分数乙醇胁迫条件下,葡萄糖质量分数占初总糖质量分数40%及以上时,果糖代谢会严重地受到葡萄糖的代谢阻遏;微通氧可有效地缩小发酵后期果糖/葡萄糖利用差异性,提高乙醇产率61.70%;较低温度明显有利于发酵后期酵母数的维持;相比外源乙醇,糖的代谢更易受到内源乙醇的影响。酒精酵母GJ2008对葡萄糖有一定偏好性,然而此种偏好性不会受到外部因素的影响。对高体积分数甘蔗汁酒精发酵而言,发酵后期需保证微通氧和较低温环境,并应尽可能减少发酵后期葡萄糖的含量,从而去除葡萄糖对果糖的代谢调控作用。     

Glycolytic sequence and respiration of Debaryomyces hansenii as compared to Saccharomyces cerevisiae

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.     

无醇啤酒酵母菌株HD34-1的生产性实验

从构建的乙醇脱氢酶(ADH)工程菌株中筛选到ADH活性最强的菌株HD34-1,以其为发酵菌株进行1吨发酵罐发酵实验,经过生产性发酵实验与理化指标的测定,证明HD34-1菌株的发酵酒精度低于1%(W/W),满足无醇啤酒的要求,并确定了该菌株啤酒发酵的发酵条件为1吨发酵罐密闭发酵,温度选择11℃,接种量为2%,糖度降为5%时终止发酵,降温至4℃封存。     

耐温絮凝酵母Saccharomyces cerevisiae KF-7的细胞活性及发酵特性研究

本文通过不同温度下对絮凝酵母ＳａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅＫＦ－７胞内储存糖含量、有机酸综合值ＯＳＲ及发酵性能的分析研究，探讨了絮凝酵母的耐温性及细胞活性。研究结果表明：酵母细胞的耐温性及细胞活性与细胞内的海藻糖含量密切相关；酵母分泌的有机酸综合值ＯＳＲ可以直观反映酵母细胞活性；在一定温度范围内，低温有利于酵母增殖，高温有利于酒精发酵；提高发酵温度可以提高酵母活性，增加出酒率。发酵试验结果表明絮凝酵母ＫＦ－７为一株性能优良菌株。     

A K2 neutral Saccharomyces cerevisiae strain contains a variant K2 M genome

K2 neutral strain Saccharomyces cerevisiae USM12 was identified and characterized. This strain carried an M double-stranded RNA (dsRNA) genome encoding for resistance to K2 toxin. The M dsRNA was larger than the K2 killer yeast M dsRNA and homoduplex analysis of denatured and reannealed K2 neurtal M dsRNA revealed an inverted duplication. Heteroduplex analysis showed that two thirds of the K2 M genome had homology with the K2 neutral M genome. Hybridization showed that the USM12 M dsRNA had significant homology with the K2 M dsRNA. Protein profiles of extracellular proteins from USM12 and a cured strain indicated that USM12 did not secrete any toxin. This is the first time that a K2 neutral yeast strain has been characterized.     

High-affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose-phosphorylating enzymes

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.