高糖胁迫对酿酒酵母抗氧化活性及代谢的影响

本文以酿酒酵母(Saccharomy cescerevisiae)为模式生物,研究了高糖培养条件对酿酒酵母的生长、抗氧化酶活性及海藻糖、甘油代谢的影响。结果表明:当葡萄糖浓度达到40%时,酿酒酵母生长的对数期延长,对酿酒酵母的生长产生了抑制作用。酿酒酵母在培养4~10 h范围内四组酿酒酵母细胞(20 g/L葡萄糖组、40 g/L葡萄糖组、60 g/L葡萄糖组和80 g/L葡萄糖组)内海藻糖的积累随着胁迫时间的增加发生显著变化(P<0.05),海藻糖的积累量呈先升高后下降的趋势,在8 h时高糖组海藻糖积累量均达到一个最高点,胞内海藻糖的浓度最高达到0.0955 mg/mL。在不同葡萄糖浓度胁迫下,酵母细胞胞内外甘油的积累随着时间增加呈现出先上升后下降的趋势,但是胞内甘油的积累量在80 g/L葡萄糖浓度时达到最高,而胞外甘油的积累量在60 g/L葡萄糖浓度时达到最高。这些结果说明在高糖胁迫下甘油和海藻糖是酿酒酵母的主要相容性溶质。另外,高糖处理后,与对照组相比,高糖组酿酒酵母胞内超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性均显著升高(P<0.05),说明这些抗氧化物酶活性物质对维持有机体胞内正常渗透压起到关键作用。该研究结果将为今后研究酿酒酵母耐高糖渗透压方面提供理论依据。     

麦麸中阿魏酸糖酯对高糖诱导HepG2细胞氧化应激的影响

研究麦麸中阿魏酸糖酯(FGs)对高糖诱导损伤的Hep G2细胞氧化应激的影响。Hep G2细胞分为正常对照组、高糖诱导损伤组、阿魏酸糖酯低剂量组(0.0565μmol/L)、阿魏酸糖酯中剂量组(0.113μmol/L)和阿魏酸糖酯高剂量组(0.565μmol/L),以荧光探针法检测细胞ROS水平,分光光度法测定细胞总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)活力,用油红O脂类染色法观察细胞脂质沉淀情况。与高糖诱导损伤组相比,FGs高、中、低剂量组细胞的ROS水平显著降低(P0.01),CAT活力显著上升(P0.01),细胞脂质沉淀量明显减少;FGs中、高剂量组细胞的GSH-PX活力显著上升(P0.01);高浓度组的T-SOD活力为(48.05±1.40)U/mg,与高糖诱导损伤组的T-SOD活力相比具有极显著差异(P0.01)。阿魏酸糖酯通过增强细胞抗氧化酶活性,抑制胞内ROS生成,抑制高糖诱导Hep G2细胞的氧化应激。     

高表达MAL62基因对面包酵母耐高糖的影响

面包酵母(Saccharomyces cerevisiae)的抗逆性对于烘焙工业至关重要。以前期获得的耐冷冻酵母突变株B+MAL62为研究对象,测定其在高糖环境下的生长特性、形态特征、胞内海藻糖与甘油的积累以及产气的变化,并与市售高糖酵母进行对比。研究发现在质量分数为40%~60%的糖胁迫环境下,B+MAL62菌株的胞内海藻糖与甘油水平分别比对照菌株提升55. 03%~64. 27%与1. 2~1. 3倍,且高糖环境下B+MAL62具有更好的细胞形态稳定性,其产气速度以及最终产气量可优于市售高糖酵母。结果表明,麦芽糖酶编码基因MAL62高表达可增强面包酵母耐高糖能力。     

硒对镉胁迫下酿酒酵母抗氧化活性的影响

利用酿酒酵母（Saccharomyces cerevisiae）作为模式生物,主要通过测定硒对镉胁迫下酿酒酵母细胞内活性氧（reactive oxygen species,ROS）和丙二醛（malondialdehyde,MDA）含量,及重要抗氧化酶包括超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）和过氧化物酶（peroxidase,POD）抗氧化活性的变化,研究硒对镉胁迫下酿酒酵母的抗氧化活性的影响。结果表明：镉处理能极显著提高酿酒酵母细胞内ROS和MDA含量（P＜0.01）,进而对酿酒酵母细胞产生氧化胁迫,在培养体系内加入一定量的外源硒可极显著（P＜0.01）增加酿酒酵母细胞内抗氧化酶SOD、POD、CAT和GSH-Px活性,提高抗氧化物谷胱甘肽含量,加强酿酒酵母细胞对ROS和MDA的清除能力,这些结果表明外源硒的添加在酿酒酵母体内亦可对镉的影响产生拮抗作用,且与在动植物体内表现出一致的趋势,后续可以利用酿酒酵母作为模式生物进一步研究硒对镉毒性拮抗作用的一些机制。     

来源于胞曲霉的α-1,2-甘露糖苷酶在酿酒酵母Δalg3Δoch1Δmnn1菌株中的定位表达

利用酿酒酵母(Saccharomyces cerevisiae)生产医药糖蛋白,必须对其N-糖基化途径进行人源化改造。在敲除酿酒酵母W303A特异性糖基化基因ALG3、OCH1和MNN1后,表达来源于胞曲霉(Aspergillus saitoi)的α-1,2-甘露糖苷酶(MsdSp)进一步改造酿酒酵母N-糖链。通过在Msd Sp的C-端添加内质网滞留信号(HDEL)和构建高尔基体滞留信号Kre2p与MsdSp的嵌合体蛋白实现MsdSp在内质网和高尔基体中的定位表达。结果表明,各突变株中均检测到Man_3GlcNAc_2和Man_4GlcNAc_2型N-糖链,并且在强启动子酵母甘油醛-3-磷酸脱氢酶启动子(GPD)控制下,内质网定位表达MsdSp的菌株KM201中,Man_3GlcNAc_2和Man_4GlcNAc_2型N-糖链含量较多。该研究为酿酒酵母N-聚糖进一步人源化改造提供依据。     

铜离子对酿酒酵母能量代谢及细胞膜功能的影响

以优良葡萄酒酿酒酵母为研究对象,采用葡萄汁模拟发酵体系,较为系统地研究了在不同浓度铜离子的作用下酵母的代谢特征。同时,从能量代谢(ATP、NADH/NAD+)、细胞膜的通透性和完整性的角度分析了发酵过程中铜离子对酵母的作用机制。结果表明,Cu~(2+)胁迫处理后葡萄糖和果糖的利用率以及乙醇的生成率降低,胞内ATP、NADH/NAD~+、核酸、蛋白质的含量以及荧光强度都比对照高。说明经Cu~(2+)胁迫处理后酵母的能量代谢受到抑制、细胞膜的完整性受损,从而导致了糖和乙醇代谢的减缓以及部分酵母菌的死亡。     

蔗糖利用型毕赤酵母工程菌的构建

巴斯德毕赤酵母(Pichia pastoris)表达系统是近20年来应用最广泛的真核表达系统之一,已有500多种蛋白在该系统得到了成功表达,然而在毕赤酵母基因组中却缺少蔗糖酶基因,因此无法利用蔗糖。本研究从酿酒酵母基因组中克隆得到蔗糖酶基因SUC2。将SUC2亚克隆后得到表达载体pPICZαA-SUC2。经电转化后,表达载体整合进入毕赤酵母KM71H基因组,得到能够利用蔗糖的毕赤酵母工程菌。     

甘露聚糖对酿酒酵母菌株生长及抗氧化活性的影响

葡萄酒酿造是一个高糖、高酸、低pH的相对恶劣生境,且发酵产生的乙醇会影响酿酒酵母(Saccharomyces cerevisiae)细胞的生长与代谢。为探究甘露聚糖在酒精发酵过程中对酿酒酵母细胞活力及抗氧化代谢的影响,以添加不同质量浓度甘露聚糖的模拟葡萄汁为试材,动态检测酒精发酵过程中酿酒酵母菌株的生物量、细胞活力及抗氧化活性。结果表明,甘露聚糖在酿酒酵母菌株生长的全过程对细胞活力具有显著提升作用(P<0.05);与对照组相比外源添加甘露聚糖能够显著提高酿酒酵母细胞的三磷酸腺苷酶(adenosine triphosphatase, ATPase)活性及细胞内还原型谷胱甘肽(glutathione, GSH)(P<0.05),可清除细胞内过量的活性氧(reactive oxygen species, ROS)、丙二醛(malondialdehyde, MDA),提高细胞的抗氧化能力。综合分析,外源添加质量浓度为300 mg/L甘露聚糖可以有效提升酿酒酵母细胞的活力及抗氧化活性。     

原花色素对酿酒酵母cAMP-PKA信号转导途径的影响

为探究酵母在氧化胁迫下外源添加原花色素对胞内海藻糖积累以及cAMP-PKA信号转导途径的影响。在氧化胁迫条件下,外加添加1g/L的原花色素,分别分析胞内海藻糖积累量、上游信号转导关键元件PKA,腺苷酸环化酶的转录规律及cAMP的水平。添加H_2O_2后再添加1 g/L的原花色素的发酵体系中酵母细胞内海藻糖产量是空白对照组的3倍,cAMP产量和腺苷酸环化酶的酶活是空白对照组的2.5倍,CYR1和BCY1基因的相对表达量较空白组均呈现明显升高趋势。cAMP会与PKA调节的亚基BCY1相结合,释放出催化亚基从而激活PKA,产生海藻糖保护细胞抵抗不利环境。该研究为以后深入探究氧化胁迫下原花色素对酿酒酵母的保护机制奠定基础,也为以后探究酿酒酵母在面临其他胁迫条件下如何抵御外界环境变化奠定基础。     

糖耗速率对浓香型白酒发酵过程异戊醇合成的影响

高级醇是白酒的风味物质之一,其含量过高影响白酒饮用的舒适度。通过考察酵母种类与初始菌浓度、发酵起始温度、大曲酶活力等因素对浓香型白酒发酵过程异戊醇合成的影响,为研究减控策略提供参考。结果表明,初始酵母种类与数量、发酵起始温度、大曲酶活力均对浓香型白酒发酵过程异戊醇合成阶段还原糖的消耗、酿酒酵母的增殖和异戊醇合成水平有影响。预估表观耗糖速率变小,酿酒酵母增殖倍数减少,异戊醇合成水平也相应降低。酿酒酵母是影响白酒发酵过程异戊醇合成的主要酵母,当发酵起始酿酒酵母数量为5×10⁷ CFU/g时,酒醅中的异戊醇比对照减少22.9%;酒醅入池发酵起始温度的降低也可显著减少异戊醇含量,降幅最高为22.6%;此外,通过降低大曲糖化酶活力也能有效减少发酵过程异戊醇的合成。该研究揭示了浓香型白酒发酵过程酿酒酵母增殖和还原糖消耗与异戊醇合成水平之间的关系,为阐明白酒发酵过程异戊醇合成机制和研究异戊醇减控策略奠定理论基础。     

HXT5 expression is determined by growth rates in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, hexose transporter (Hxt) proteins transport glucose across the plasma membrane. The Hxt proteins are encoded by a multigene family with 20 members, of which Hxt1-4p and Hxt6-7p are the major hexose transporters. The remaining Hxt proteins have other or unknown functions. In this study, expression of HXT5 under different experimental set-ups is determined. In glucose-grown batch cultures, HXT5 is expressed prior to glucose depletion. Independent of the carbon source used in batch cultures, HXT5 is expressed after 24 h of growth and during growth on ethanol or glycerol, which indicates that growth on glucose is not necessary for expression of HXT5. Increasing the temperature or osmolarity of the growth medium also induces expression of HXT5. In fed-batch cultures, expression of HXT5 is only observed at low glucose consumption rates, independent of the extracellular glucose concentration. The only common parameter in these experiments is that an increase of HXT5 expression is accompanied by a decrease of the growth rate of cells. To determine whether HXT5 expression is determined by the growth rate, cells were grown in a nitrogen-limited continuous culture, which enables modulation of only the growth rate of cells. Indeed, HXT5 is expressed only at low dilution rates. Therefore, our results indicate that expression of HXT5 is regulated by growth rates of cells, rather than by extracellular glucose concentrations, as is the case for the major HXTs. A possible function for Hxt5p and factors responsible for increased expression of HXT5 upon low growth rates is discussed.     

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.     

TOR kinase pathway and 14-3-3 proteins regulate glucose-induced expression of HXT1, a yeast low-affinity glucose transporter

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14-3-3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14-3-3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF-Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B-subunit, in the glucose induction of HXT1 is also discussed.     

Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.     

Sugar consumption and ethanol fermentation by transporter-overexpressed xylose-metabolizing Saccharomyces cerevisiae harboring a xyloseisomerase pathway

Four kinds of transporters, HXT1 and HXY7 from Saccharomyces cerevisiae, and GXF1 and GXS1 from Candida intermedia, were overexpressed in xylose-metabolizing S. cerevisiae harboring a xyloseisomerase-based pathway. Overexpression of transporter enhanced sugar consumption and ethanol production, and GXF1 was efficient for ethanol fermentation from both glucose and xylose.