Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

Analysis of the Involvement of Different Ceramide Variants in the Response to Hydroxyurea Stress in Baker's Yeast

Sphingolipids have been identified as important signaling compounds in stress responses. However, it is not always clear how different sphingolipid profiles are achieved in a particular stress situation. Here we propose a detailed mass action model, containing 42 dependent variables and 137 reactions, that offers explanations of the roles of variant ceramides species, which differ in the lengths of their fatty acyl chains and their saturation state, in the response to hydroxyurea stress. The simulations demonstrate that the cells manage to achieve hydroxyurea tolerance through a well-coordinated, differential usage of the variant ceramide species. Moreover, the results suggest that key enzymes have different affinities toward saturated and unsaturated fatty acyl chains, which implies that the saturation state affords the cells with an additional mode of regulation that had not been recognized so far. These conclusions from our computational analysis are yet to be validated experimentally.     

刚毛柽柳ThDREB基因在酵母中的表达及抗逆能力分析

DREB蛋白能特异地与DRE（dehydration responsive element）顺式作用元件结合,从而调控下游多个与逆境相关基因的表达,在植物对多种逆境胁迫的应答反应中起重要调节作用。为了研究刚毛柽柳ThDREB基因是否具有抗逆功能,将ThDREB基因插入到酵母表达载体pYES2中构建成重组载体,转入酿酒酵母（Saccharomyces cerevisiae）中获得重组型酵母。分别比较转ThDREB基因酵母和转空载体对照酵母在山梨醇、H₂O₂、CdCl₂、NaCl、Na₂CO₃、MgCl₂、-20℃胁迫处理之后的存活能力。结果显示,ThDREB基因能有效提高转基因酵母的抗干旱、盐、碱、氧化、重金属及低温胁迫的能力,表明ThDREB基因可能参与了柽柳多种抗逆调控过程。     

Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATα MAL1 MAL3g SUC2 SUC4 gall. The MAL1 gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains.     

Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATalpha MAL1 MAL3g SUC2 SUC4 gall. The MAL1 gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains.     

胞红蛋白酵母双杂交载体的构建与表达

胞红蛋白（CGB）是一种新发现的分布于胞浆与胞核的携氧珠蛋白。为探讨CGB在体内的相互作用蛋白,从而促进对其分子调控网络的认识,根据CGB基因的开放阅读框架设计并合成PCR引物,从人胎肝cDNA文库中扩增得到该基因编码区.测序分析正确后将其定向克隆到酵母表达载体pGBKT7中,构建获得CGB的酵母表达载体pGBKT7-CGB,并在酵母菌AH109中表达。提取酵母总蛋白并利用标签蛋白（myc）的抗体进行免疫印迹检测。结果表明所构建的CGB酵母表达载体能够在酵母中高效表达,可用于后续的酵母双杂交文库的筛选工作。     

人脂素基因LIPIN1在酵母中的异源表达及细胞功能分析

脂类代谢调控是维持生物体能量平衡的重要环节,脂类代谢调控的紊乱与肥胖症、糖尿病和高血压等疾病密切相关。脂素基因三LIPIN1是诱导脂肪细胞分化、调控脂类合成的关键基因．其编码的磷脂磷酸酶（phosphatidate phosphatase,PAP）在人体三酰甘油合成中起关键作用,是维持人体脂类平衡的重要保障。此外,该基因还作为重要的转录辅激活因子参与多种生长及营养代谢调控。多种生物中均有类似功能的基因被发现,暗示了其功能的多样性及物种间的保守性。该文利用酿酒酵母在脂类代谢研究中性状易于表征、同源基因剧刖功能明确的优势,通过同源重组技术构建脂素缺陷型酵母,探索脂素基因在维持酵母正常生长及脂类合成中的重要作用,并通过功能互补及生物信息学技术对比分析了人源LIPIN1基因与酵母PdH1基因编码蛋白在结构和功能上的保守性,为脂素基因LIPIN1的细胞功能研究提供基础数据。     

二色补血草LbDREB基因在酵母中的抗逆性分析

利用酵母表达系统研究了二色补血草的DREB基因（LbDREB）对不同胁迫的抗性。将LbDREB构建到酵母表达载体pYES2中,转化到酿酒酵母INVSc1菌株中,并以转空pYES2质粒的酵母INVSc1（pYES2）作为对照,通过比较两种酵母在不同胁迫下的存活率来研究LbDREB基因对NaCl、KH_2PO_4、Na_2CO_3、NaHCO_3、低温、干旱、CuSO_4和CdCl_2胁迫的抗性。结果表明,LbDREB转化的酵母在各种胁迫下的存活率均明显高于转空pYES2的对照酵母,说明LbDREB基因除了具有传统认为的抗旱、耐盐、抗寒的作用外,还具有抗KH_2PO_4、Na_2CO_3、NaHCO_3、CuSO_4和CdCl_2等胁迫的能力。     

Global Gene Expression Analysis of Yeast Cells during Sake Brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

Expression of the Maize Mnsod (Sod3) Gene in Mnsod-Deficient Yeast Rescues the Mutant Yeast under Oxidative Stress

Superoxide dismutases (SOD) are ubiquitous in aerobic organisms and are believed to play a significant role in protecting cells against the toxic, often lethal, effect of oxygen free radicals. However, direct evidence that SOD does in fact participate in such a protective role is scant. The MnSOD-deficient yeast strain (Sod2d) offered an opportunity to test the functional role of one of several SOD isozymes from the higher plant maize in hopes of establishing a functional bioassay for other SODs. Herein, we present evidence that MnSOD functions to protect cells from oxidative stress and that this function is conserved between species. The maize Sod3 gene was introduced into the yeast strain Sod2d where it was properly expressed and its product processed into the yeast mitochondrial matrix and assembled into the functional homotetramer. Most significantly, expression of the maize Sod3 transgene in yeast rendered the transformed yeast cells resistant to paraquat-induced oxidative stress by complementing the MnSOD deficiency. Furthermore, analyses with various deletion mutants of the maize SOD-3 transit peptide in the MnSOD-deficient yeast strain indicate that the initial portion (about 8 amino acids) of the maize transit peptide is required to direct the protein into the yeast mitochondrial matrix in vivo to function properly. These findings indicate that the functional role of maize MnSOD is conserved and dependent on its proper subcellular location in the mitochondria of a heterologous system.     

面包酵母不对称还原酮基泛解酸内酯

目的:利用市售面包酵母作催化剂,对酮基泛解酸内酯不对称还原进行了研究,并考察了底物浓度、辅因子、环糊精添加量和pH值对不对称还原反应的影响.方法:以市售面包酵母作催化剂,酮基泛解酸内酯作为底物在摇瓶中进行催化,用手性色谱柱对催化产物进行了检测.结果:发现反应条件为底物浓度低于2.56-mg/ml,辅因子选用蔗糖,β-环糊精添加量为16.7mg/ml,pH 4～5适合于酮基泛解酸内酯的不对称还原.在如上优化的催化条件下,产物得率大于45%,对映体过量率(e.e.)大于90%.结论:以酵母细胞作催化剂进行酮基泛解酸内酯不对称还原工艺具有生产光学纯泛解酸内酯的潜力.     

利用枣汁生产面包酵母

面包酵母是一类用来提高面包质量的重要添加剂。目前,不同国家主要采用分批培养、补料分批培养或连续培养的方式来生产面包酵母。酿酒酵母是用来发酵面团中淀粉的理想微生物,除了提升食品香味,增加口感之外,这一发酵过程可以产生多种维生素和蛋白质。用于生产酵母生物量的主要成分包括各种碳源,如甜菜糖蜜和甘蔗糖蜜等。由于甜菜糖蜜可用于高产率地生产乙醇,加之其带来的生物环境污染和废水处理问题,因此需要考虑用其他糖类来生产面包酵母。其中一个代替性糖源是枣。由于各种原因,伊朗每年都浪费大量的枣。研究了用枣作为培养基碳源的可行性。将废枣榨成汁,然后研究了酵母的产量和生长速率。结果发现,在pH3.4,温度30℃,通风量1.4vvm,发酵罐搅拌速度500r/min时,酵母对底物的产率接近50%。     

转甲状腺素蛋白基因在酵母中的表达

用限制酶BamHI将含人TTR基因的DNA片段(约493bp)从质粒 pSK-TTR上切下后并插入到分泌型载体pHIL-SI的BamHI位点上,经酶切鉴定重组质粒pHIL-SI-TTR上的TTR基因插入方向正确。将pHIL-SI-TTRDNA用BglII酶切后,转入真核表达系统——毕赤氏酵母。得到的转化子经摇瓶发酵,上清液用15%SDSPAGE检测,有TTRF的表达。经DEAESepheroseF.F离子交换柱和SephacrylS200分子筛层析,得到纯化的TTRF。体外实验表明TTRF对肝癌细胞有抑制作用。     

醇母PHO81基因的克隆和表达

从酵母染色体ＤＮＡ中获得１个约３．０ｋｂ的ＢａｍＨＩ的克隆片段和１个约５．０ｋｂ的ＰｓｔＩ片段,前者包含１７４５ｂｐ的ＰＨＯ８１基因编码序列及１２４４ｂｐ的上游序列,后者包含２２３６ｂｐ的编码序列及约２．８ｋｂ的下游序列,经拼接得到完整的ＰＨＯ８１基因。以ＵＲＡ３基因取代部分ＰＨＯ８１的编码序列,通过体内同源重组,获得ＰＨＯ８１基因缺陷的酵母细胞株。构建ＰＨＯ８１－ＬａｃＺ融合基因,以β－半乳糖苷酶的活力表示ＰＨＯ８１基因的表达水平,研究了它的表达作用。ＰＨＯ８１基因为阻遏型表达,受无机磷浓度的控制,高磷使基因表达阻遏,低磷去阻遏。ＰＨＯ８１对其自身的表达有正调控作用,它与ＰＨＯ５和ＰＨＯ１１基因的调控模式相似,但ＰＨＯ８１上游调控序列和ＰＨＯ５及ＰＨＯ１１的同源性很低。     

小麦非生物胁迫相关基因TaAIM的表达分析

MYB转录因子是一个在植物的应激反应中起着核心作用的蛋白质家族.为了进一步研究小麦非生物胁迫诱导转录因子的表达特性,利用同源克隆的方法从小麦中获得了 1个R2R3-MYB转录因子基因TaAIM,采用生物信息学方法对TaAIM的序列进行分析,采用半定量RT-PCR方法研究TaAIM在不同组织以及不同非生物逆境胁迫条件下的...     

Differentiated Gene Expression in Cells within Yeast Colonies

Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains. With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development. We used the library of S. cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter. Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated. We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2. S. cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain. The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.     

Self-Cloning Baker's Yeasts That Accumulate Proline Enhance Freeze Tolerance in Doughs

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding γ-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.