MSN4基因的过表达对酿酒酵母耐受性的影响研究

以实验室现有菌种AY12a为出发菌株,URA3基因作为筛选标记,利用胞内重组,在MSN4基因的N端加上强启动子PGK1p以实现基因的过表达,最终通过多聚酶链式反应（PCR）验证,成功构建突变株AY12a-msn4。结果表明,该突变株具有一定的耐高温性能,在55 ℃条件下热击后仍能正常生长。同时将突变株AY12a-msn4与出发菌株AY12a进行玉米高温浓醪发酵,并测定发酵完成后的酒精度、残糖、48 h细胞存活率、CO2失重及发酵时间。结果表明,突变株AY12a-msn4发酵液酒精度提高3.85%,48 h细胞存活率上升,残糖含量下降14.5%。     

Understanding the mechanism of heat stress tolerance caused by high trehalose accumulation in Saccharomyces cerevisiae using DNA microarray

DNA microarray analysis was performed to examine the stress tolerance mechanism of a Saccharomyces cerevisiae recombinant strain exhibiting high trehalose accumulation and heat stress tolerance. Results suggest that the upregulation of sugar transporter genes is one of the key events for heat stress tolerance of the recombinant strain.     

酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响

以sod1Δ、sod2Δ、sod1Δsod2Δ酿酒酵母基因缺失菌株为遗传材料,采用休止细胞梯度生长法,分析SOD1和SOD2基因缺失对高温、乙醇毒性、高渗透压、高盐、乙酸毒性及营养饥饿胁迫条件耐受性的影响。结果显示,与野生型菌株相比,sod1Δ菌株对高温、高渗透压和乙酸胁迫的耐受性降低;sod2Δ菌株耐受性无明显变化;sod1Δsod2Δ双缺失菌株对高温、乙醇毒性、乙酸毒性、高渗透压和高盐的耐受性均下降,表明酵母超氧化物歧化酶基因与多种胁迫耐受性密切相关。     

高耐性酿酒酵母的筛选及其耐受性研究

优良的耐逆性菌株的添加能有效提高酱油生产效率和产品品质风味,该研究筛选出两株耐高温、耐高渗和耐高酸的酵母菌株用于酱油发酵。通过对酵母菌株高温热激之后稀释点板,对比各稀释度的菌落数量和形态,以及通过在高渗板和高酸板上各个菌的生长情况和在抗性培养基中菌的生长曲线测定来对比各菌株的耐受性。稀释点板实验以及生长曲线结果都显示,酿酒酵母L-19和L-38在55 ℃热激条件下以及在分别含有6%NaCl、0.6%乙酸和5%乳酸固体平板上菌落形态和大小都优于酱油酵母,而且在含有高盐和高酸的液体培养基中生长速率均高于酱油酵母。因此,成功筛选出两株具有高耐性的酿酒酵母。     

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。     

Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.     

Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae

To examine the effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae, we constructed deletion strains of all combinations of the trehalase genes ATH1, NTH1 and NTH2 and examined their growth behaviour and intracellular trehalose accumulation under non‐stress and saline‐stress conditions. Saline stress was induced in yeast cells by NaCl addition at the exponential growth phase. All deletion strains showed similar specific growth rates and trehalose accumulation to their parent strain under non‐stress conditions. However, under the saline stress condition, one single deletion strain, nth1Δ, two double deletion strains, nth1Δ ath1Δ and nth1Δ nth2Δ, and the triple deletion strain nth1Δnth2Δ ath1Δ, all of which carry the nth1Δ deletion, showed increased trehalose accumulation as compared to the parent and other deletion strains. In particular, our statistical analysis revealed that the triple deletion strain showed a higher growth rate under the saline stress condition than the parent strain. Moreover, some deletion strains showed further trehalose accumulation under non‐stress conditions by overexpression of the TPS1 or TPS2 genes encoding the enzymes related to trehalose biosynthesis at the mid‐exponential phase. Such increased trehalose accumulation prior to NaCl addition could improve the growth of these strains under saline stress. Our results indicate that high trehalose accumulation prior to NaCl addition, rather than after NaCl addition, is necessary to achieve high growth activity under stress conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.     

Reactive oxygen species may influence the heat shock response and stress tolerance in the yeast Saccharomyces cerevisiae

Moderate levels of reactive oxygen species (ROS) have been implicated as second messengers in a number of biochemical pathways, and in animal cells have been associated with the activation of the heat shock response (HSR). Here, using an intracellular probe, we demonstrate that differential accumulation of ROS in the yeast Saccharomyces cerevisiae is strongly associated with differential induction of an HS reporter gene over a range of heat shock temperatures. There was a good correlation between cellular ROS levels and the levels of HS-induced reporter gene expression between 37 degrees C and 44 degrees C, both reaching maximal values at 41 degrees C. Furthermore, the addition of 150 microM H2O2 to the yeast cells during heat treatment resulted in a 3 degrees C decrease in the temperature required for maximal induction of the HS expression vector--an increased HS sensitivity that corresponded to a concomitant increase in ROS levels at these lower HS temperatures. Conversely, cells treated with 10 mM of the antioxidant ascorbic acid required a temperature that was 2 degrees C above that required in untreated controls for maximal induction of the HS expression vector. This decreased HS sensitivity corresponded to a decrease in ROS levels at these higher HS temperatures. Finally, cell viability assays reveal that intrinsic thermotolerance remains high in control cells despite concomitant decreases in HS-reporter gene expression and ROS accumulation between 41 degrees C and 44 degrees C. We conclude that the sensitivity of the yeast HSR is strongly associated with ROS accumulation, and suggest that ROS-mediated signalling ensures cooperation between the HS and the antioxidant responses.     

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with high mitotic stability. The pMIRY integrative vector system, based upon targeted integration into the yeast rDNA locus, developed in our laboratory satisfies these criteria. However, insertion of a (foreign) gene drastically reduced its mitotic stability of the resulting vector in comparison to its parent. In this paper we have investigated a number of possible reasons for this reduction in stability. The results demonstrate that plasmid size is an important, but not the only, determinant of mitotic stability. Stable maintenance is only observed when the complete plasmid has a size no larger than that of the rDNA unit (9·1 kb). In addition stability depends upon the nature of the rDNA fragment present in the plasmid, required for targeting its integration. On the other hand, it turned out to be irrelevant for mitotic stability whether the heterologous gene was expressed or not. These findings will be important in the design of a pMIRY vector suitable for industrial production of heterologous proteins.     

微卫星在酿酒酵母研究中的应用

微卫星因其在真核生物基因组中分布广泛、多态性丰富、呈选择中性和共显性遗传等特点,已成为继RFLP之后的第2代分子遗传标记,广泛用于酿酒酵母菌株的分类鉴定、菌株间的亲缘关系分析和酿酒酵母群体遗传多样性分析。本文综述微卫星标记的应用原理及其在酿酒酵母研究中的应用进展。     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

GIS1基因对酿酒酵母耐受性的研究

该研究主要通过过表达GIS1基因来提高酿酒酵母的耐受性。在GIS1基因的N端加入强启动子PGK1p来实现GIS1基因的过表达,然后通过稀释点板实验来对比其对热激、乙醇、渗透压以及乙酸的耐受性,同时通过高温生长曲线和高温浓醪发酵测定其高温耐受性。结果表明,在相同的稀释倍数下,55 ℃热击4 min之后菌株AY12a-gis1的生长能力明显优于出发菌株,在含有5%（V/V）乙酸的平板上改造菌和出发菌耐受性相差不大,同时通过38 ℃浓醪发酵实验发现菌株AY12a-gis1的酒精度提高了3.79%,残糖略有下降,且发酵时间与亲本菌株相一致。因此通过过表达GIS1基因得到菌株AY12a-gis1,是对工业乙醇发酵有一定应用价值的优良耐逆性菌株。     

低氮对酿酒酵母发酵过程中酯类物质合成的影响

采用顶空固相微萃取-气相色谱-质谱法（HS-SPME-GC-MS）测定了不同程度低氮胁迫下的模拟葡萄汁酒精发酵过程中酯类物质的含量。结果表明,低氮并没有改变各种酯类物质的生成规律,但影响了其发酵结束时酯类物质的含量。在发酵过程中,乙酸乙酯、己酸乙酯、辛酸乙酯和癸酸乙酯的含量逐渐增加;丁酸乙酯的含量在发酵第8天达到最大值后逐渐降低;乙酸异戊酯的含量先增加后在发酵第12天时达到最低值后又迅速增加;乙酸苯乙酯的含量在发酵前期（发酵4～6 d）迅速达到一个峰值,随后其含量缓慢降低;低氮胁迫下酯类物质的总量降低,其中乙酸苯乙酯和癸酸乙酯的含量增加,乙酸乙酯、乙酸异戊酯、丁酸乙酯和己酸乙酯的含量降低。因此,在葡萄酒的生产过程中可适当提高氮源浓度来提高酯类物质含量。     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株.在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失.因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选...     

Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae

We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.     

发酵工业中酿酒酵母耐性机制的研究进展

由于工业生产条件和成本的限制,发酵过程中酿酒酵母会经受多种不同环境胁迫因子的影响。研究不良环境中酿酒酵母的耐性机制,旨在为提高产品质量、降低生产成本以及相应的基因改良提供理论依据。文中主要综述了酿酒酵母耐受高渗、氧化和乙醇等胁迫因素的机理以及海藻糖对酵母细胞的保护作用。