The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation

We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions. A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions. No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain. The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation. However, these two carriers have different functions. The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone. The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation. The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation. These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation. This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle.     

The low-affinity component of the glucose transport system in Saccharomyces cerevisiae is not due to passive diffusion

It has been claimed that the low-affinity component of glucose transport in Saccharomyces cerevisiae is due to passive diffusion of the sugar across the plasma membrane. We have investigated this possibility. For this purpose we have measured the permeability coefficient of hexoses in this organism. We have found that this coefficient is at least two to three orders of magnitude lower than required to account for the low-affinity component of glucose transport, and have concluded that this component is not due to passive diffusion.     

酿酒酵母的糖转运机理研究进展

简要概述了酿酒酵母所利用的 3种主要糖 :葡萄糖、麦芽糖和麦芽三糖转运进入细胞机理的研究现状 ,分析了目前存在的问题 ,提出了今后的研究方向。     

α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的策略

综述了宿主细胞的表达特点，外源基因的来源，表达载体和转化方法对α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的影响，并在此基础上提出了相应的策略。     

Galactose inhibition of the constitutive transport of hexoses in Saccharomyces cerevisiae

The relationship between the pathways of glucose and galactose utilization in Saccharomyces cerevisiae has been studied. Galactose (which is transported and phosphorylated by inducible systems) is a strong inhibitor of the utilization of glucose, fructose and mannose (which have the same constitutive transport and phosphorylation systems). Conversely, all these three hexoses inhibit the utilization of galactose, though with poor efficiency. These cross-inhibitions only occur in yeast adapted to galactose or in galactose-constitutive mutants. The efficiency of galactose as inhibitor is even greater than the efficiencies of each of the other three hexoses to inhibit the utilization of each other. Phosphorylation is not involved in the inhibition and the transport of sugars is the affected step. The cross-inhibitions between galactose and either glucose, fructose or mannose do not implicate utilization of one hexose at the expense of the other, as it occurs in the mutual interactions between the latter three sugars. It seems that, by growing the yeast in galactose, a protein component is synthesized, or alternatively modified, that once bound to either galactose or any one of the other three hexoses (glucose, fructose or mannose), cross-interacts respectively with the constitutive or the inducible transport systems, impairing their function.     

乙醇胁迫对啤酒酵母生长及蛋白表达的影响

研究了乙醇胁迫对啤酒酵母生长的影响,应用光镊拉曼光谱(LTRS)技术获得并分析酵母单细胞拉曼光谱,从分子水平分析酿酒酵母细胞内的蛋白质组变化。结果表明乙醇可抑制酵母生长,随着乙醇浓度的提高,酵母细胞直径变小、稳定期推迟、生物量和蛋白质含量也呈减少趋势;通过光镊拉曼光谱分析可了解酵母细胞内的乙醇浓度和生化组成的相对含量等信息;在不同乙醇浓度下,采用SDS变性凝胶电泳(SDS-PAGE)共检测到22个明显的差异条带,并对其中7个差异条带进行质谱鉴定,发现这7个差异蛋白的功能主要与端粒稳定性、细胞自溶及代谢相关;不同乙醇浓度可诱导酵母特定蛋白质表达发生变化,如HSP104等蛋白质,说明这些蛋白质所参与的代谢途径在啤酒酵母乙醇耐性中具有普遍作用。     

Expression of recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae.

A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.     

The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat

The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C_18:0 fatty acids in total phospholipids at the expense of C_16:0 fatty acids. The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids. The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S. cerevisiae. It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S. cerevisiae cells.     

Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae

A dextranase-encoding cDNA from L. starkeyi KSM22 was isolated and characterized. The 2052 bp cDNA fragment (lsd1) harbouring the dextranase gene exhibited one open reading frame (ORF) composed of 1824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR, including a 27 bp poly(A) tail. The lsd1 gene contains no introns. The open reading frame encodes a 608 amino acid polypeptide (LSD1) with a 67.6 kDa predicted molecular mass. There was a 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of LSD1 dextranase exhibits distant similarity with the enzymes of the glycosyl hydrolase family 49 that comprises Penicillium dextranase. The optimum pH of LSD1 was 6.0 and the optimum temperature was 37 degrees C. LSD1 dextranase activity was substantially abolished by exposure to 1 mM Hg2+, Ag3+ and Mn2+. LSD1 exhibited high hydrolysing activity towards dextran (100%), soluble starch (22%) and mutan (8%).     

高压静电场对酿酒酵母菌的作用研究

研究了高压静电场对酿酒酵母菌的作用,实验结果表明,高压静电场对酿酒酵母菌细胞的生长速率有影响。不同的电场强度、作用时间对细胞生长的速率有所不同。此外还分别从物理学、生物学的角度研究了高压静电场作用于酿酒酵母菌的微观机理。     

A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.

A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.     

分光光度法测定酿酒酵母细胞悬液浓度研究

采用分光光度法测定酿酒酵母(Saccharomyces cerevisiae)在不同生长条件、不同时间菌悬液OD600nm值,建立OD600nm在不同稀释区间的分段回归方程,利用OD600nm回归值绘制生长曲线,对比平板菌落计数法测得的生长曲线,研究在对数期与稳定期,OD600nm值与菌体数量以及稀释倍数的关系。结果表明,稀释前后OD600nm值在对应区间内呈线性关系,用OD600nm回归值绘制的生长曲线没有明显的衰亡期,其主要是由于在对数期以后OD600nm值与菌体数量不呈线性关系,而在稳定期以前两者线性良好;得到了OD600nm值在0.2~0.8范围内与细胞数量的对应关系,还发现实际OD600nm值与稀释倍数之间并不是简单的乘积关系,而呈幂函数关系。     

Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae

5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues.     

瑞氏木霉内切葡聚糖酶基因在酿酒酵母中的表达研究

将PCR合成的瑞氏木霉 (Trichodermareesei) β 内切葡聚糖酶I(EGI)cDNA基因片断分别插入酵母met1 0和 pgk1启动子和终止子序列之间 ,构建了在不同启动子控制下 ,具有不同拷贝数的eg1表达分泌质粒pRS41 5ME、pRS41 5PE和 pRS42 5PE。通过电转化使重组质粒转移至实验室酿酒酵母H1 5 8菌株中 ,分别得到了 3株酵母转化子H1 p、H2p和H1m。在 3株酵母转化子中 ,重组 β 内切葡聚糖酶I都能在酶自身信号肽序列引导下进行分泌型表达。在YPD培养基中 3株重组酵母生长速率大致相同 ,H1m ,H1 p与H2 p的内切葡聚糖酶活力分别为 70 .4,1 2 6.7和 1 2 5 .0U/mL。     

肉桂醛诱导酿酒酵母Persisters细胞的形成

本文研究了肉桂醛对酿酒酵母persister细胞形成的影响。通过96孔板微量法测定肉桂醛对酿酒酵母生长的最小抑制浓度(MIC)为0.4 m M;采用流式细胞仪以及梯度稀释滴平板计数法研究了肉桂醛处理后酿酒酵母persister细胞的形成情况。结果表明,肉桂醛可以抑制酿酒酵母生长,且能够诱导酿酒酵母细胞形成persister状态,该状态下的细胞对两性霉素B产生耐药性。进一步研究发现肉桂醛处理后可以使酿酒酵母细胞停滞在细胞周期的任何阶段,而雷帕霉素诱导的细胞自噬只能停留在G1期,所以酿酒酵母perisister与自噬状态存在区别。目前,对于Persister的研究集中在原核微生物,对真核生物persister的研究非常有限。由于persister群体通常占总群体极小一部分,这就给基因水平上研究persister的形成机制带来很大的挑战。本研究发现肉桂醛处理酿酒酵母细胞后,可以促使其大部分细胞形成persister。这就为从基因水平上认识真核生物persister的形成机制提供了方法,实验结果表明YGL基因也与酿酒酵母persister的形成有很大关系。