酿酒酵母SNF4基因敲除缺失菌株的构建

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件,转化到酿酒酵母YS2获得阳性克隆子,然后将质粒pSH65转到阳性克隆子中,半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记,获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明,缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统,成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。     

Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae

Abstract The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1 . Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11 . The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3 ) that, however, was unable to suppress other FLO1 genes in other flocculent strains.     

酿酒酵母基因编辑技术研究进展

酿酒酵母Saccharomyces cerevisiae是代谢工程中最重要的宿主之一,先进的基因编辑技术已经被广泛应用于酿酒酵母细胞工厂的设计和构建.随着基因编辑技术的飞速发展,早期基于重组酶和同源重组的基因编辑技术逐渐被新型基因编辑系统所替代.文中对酿酒酵母基因编辑技术的原理和应用进行了总结,包括经典的酿酒酵母基因编...     

PGA基因在酿酒酵母中的表达研究

本文根据GenBank 中巨大芽孢杆菌（Bacillus megaterium）的PGA基因序列设计了上下游引物,通过PCR扩增出巨大芽孢杆菌1.1741中的PGA基因。将该基因连接到T7lac启动子控制下的表达载体pYES2(amp+,ura+)上,构建了重组质粒pYES2-PGA。用LiAc/SSDNA/PEG方法将其转化进酿酒酵母(Saccharomyces cerevisiae)H158中表达,在不需要苯乙酸诱导的重组菌株发酵液中检测到了青霉素酰化酶活性,最高酶活达到0.75 U/ml。将该PGA基因测序结果与GenBank中巨大芽孢杆菌L04471.1、U07682.1和Z37542三株的PGA基因序列比对,表现出很高的同源性,分别达到97.1%、99.8% 和99.8%。     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

酿酒酵母ADH3基因的敲除

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。     

Improved method for the PCR-based gene disruption in Saccharomyces cerevisiae

The PCR-based gene disruption strategy originally devised by Baudin et al. is widely used for gene targeting in Saccharomyces cerevisiae. An advantage of this strategy is its simplicity in making targeting constructs. The efficiencies of the targeted disruption are highly variable from locus to locus, however, and often very low. In this report, a method for improving the gene deletion efficiency is described.     

酿酒酵母进级进化过程中的基因丢失现象初探

酿酒酵母进级进化过程中的基因丢失问题很少被研究．利用现有的蛋白质数据库，通过设定判断标准，预期可以获得更多这方面的案例以便进行相关问题的研究．利用SMART蛋白质域家族数据库，根据在已完成测序的7种真核生物蛋白质域家族成员中存在或不存在酿酒酵母蛋白质域信号，判断是否发生了基因丢失．通过这种方法，共有6个酿酒酵母基因判断为在进级进化过程中发生了丢失，还探讨了这些基因发生丢失的可能机制．     

甲磺酸甲酯对酵母DNA损伤及端粒酶活性的影响

观察甲磺酸甲酯 (MMS)对酿酒酵母S2 88C细胞染色体DNA的损伤及端粒酶活性的调节。结果表明 ,甲磺酸甲酯引起酵母细胞DNA损伤 ,随着MMS浓度的增加及作用时间的延长 ,DNA损伤程度加重 ,同时明显提高酵母细胞端粒酶活性。当用 0 .4mmol/LMMS作用 72h后 ,端粒酶活性最高 (是对照组的1.4 7倍 ) ,在作用 96h及 12 0h后端粒酶活性逐渐下降 ,但均高于对照组。甲磺酸甲酯对酿酒酵母S2 88C细胞端粒酶活性的上调作用可能与其DNA损伤有关 ,断裂DNA的损伤后修复可能是端粒酶介导的。     

WEB2基因在酿酒酵母S期检查点通路上调控RNR3基因表达

WEB2基因参与酿酒酵母S期检查点调控机制,而RNR3基因位于该调控通路末端,DNA损伤或合成阻断时,S期检查点通路诱导RNR3过度表达。因此,通过确定WEB2在该检查点通路上是否参与调控RNR3基因的表达,将有助于进一步明确WEB2基因在检查点通路上的工作位点,了解WEB2基因如何发挥检查点调控功能。构建RNR3-LacZ基因融合质粒,用于检测酵母细胞内RNR3基因的诱导性。诱导性可以通过测定β-半乳糖苷酶的活性而得知。利用DNA损伤药物甲磺酸甲酯(MMS)及DNA合成阻断剂羟基脲(HU)处理酵母细胞,测定WEB2基因突变株和野生株细胞内RNR3基因的诱导性。结果,WEB2突变株细胞中诱导活性分别增加(8.27±0.38)倍和(9.55±0.24)倍,而野生株分别增加了(83.32±2.42)倍和(124.67±2.87)倍。反映RNR3基因在WEB2突变株中的诱导性低于野生株。同RAD53突变株相比,后者的RNR3基因的诱导性更低,仅为(2.37±0.18)倍和(2.91±0.13)倍。说明WEB2基因突变影响S期检查点通路的信号传递至RNR3基因,所以在酿酒酵母S期检查点通路上,WEB2工作在RNR3基因上游,参与调控RNR3的表达,但调控能力不如RAD53基因强。     

酿酒酵母基因组位置效应对外源基因表达的影响

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程,而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响,以绿色荧光蛋白为报告基因,通过双交换同源重组方法,对酿酒酵母单基因敲除库进行高通量转化,构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析,构建高表达位点库和低表达位点库,共发现促进绿色荧光蛋白表达的位点428个,抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布,从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考,对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。     

Detection of inactive precursors of beta-glucanases in Saccharomyces cerevisiae

Accumulation and secretion of beta-glucanases have been studied in vivo by using a thermosensitive secretory mutant of Saccharomyces cerevisiae blocked at the endoplasmic reticulum level (sec 18-1). When incubated at the restrictive temperature no accumulation of active glucanases was observed. Following a shift to permissive conditions in the presence of cycloheximide a rise in the internal activity took place. The increase in total glucanase activity was partially due to the activation of an exo-glucanase that hydrolyzes PNPG. It is concluded that glucanases are synthesized in inactive precursor forms and are converted to the active forms in their secretory pathway.     

HBV preS/S基因克隆及其在酿酒酵母中的表达

目的:探索利用酿酒酵母系统表达乙型肝炎病毒(HBV)preS／S基因。方法:利用PCR技 术,以HBV病毒DNA为模板,体外扩增HBV preS／S基因。然后构建重组表达载体pESC-preS／S。 用LiAc法转化酿酒酵母YPH50,选取重组菌进行培养,并诱导表达外源蛋白。提取蛋白浓缩后 进行SDS-PAGE分析,并经Western blot分析鉴定。结果:实验结果表明重组菌能够表达HBV preS／S蛋白。结论:利用酿酒酵母系统可成功表达HBV preS／S基因,为制备新型预防性疫苗提供 条件。     

Detection and Characterization of Fungal-Specific Proteins in Saccharomyces cerevisiae

In this study, I searched for fungal-specific proteins in the genome of the budding yeast Saccharomyces cerevisiae, inferred from a comparison of amino acid sequences. I used the GTOP (Genomes to Protein structures and functions) database of the DDBJ (DNA Data Bank of Japan), which consists of 21 genomes from Archaea, 203 genomes from Bacteria, and 50 genomes from Eucarya (including 18 fungal genomes). Among 5,874 proteins of S. cerevisiae, 1,551 have homologs only in Eucarya, and 504 of the 1,551 have homologs only in fungi. To find fungal-specific proteins, homologs of the homologs have been searched repeatedly. As a result, 132 of the 504 are characterized as fungal-specific proteins. The genes encoding the 132 fungal-specific proteins are not included in the list of essential genes for viability in the S. cerevisiae genome deletion project. Among the 132 proteins, 99 are S. cerevisiae-specific, and no protein that is distributed among 10 or more of the 18 fungal species exists. In addition, most of the fungal-specific proteins are very small and functionally unknown. My results show that the fungal-specific proteins have short evolutionary histories, suggesting that S. cerevisiae produces novel proteins and that ancestral fungi also produced small proteins most of which have disappeared or have been combined with other proteins during fungal evolution.     

热水提取酿酒酵母中S-腺苷-L-甲硫氨酸的研究

研究了采用热水法直接提取酿酒酵母胞内产物S-腺苷-L-甲硫氨酸（SAM）北京,并且通过实验对影响SAM抽提的5个条件：抽提温度、抽提时间、热水用量、搅拌转速、硫酸用量等进行了优化。得出的最佳实验条件为：温度70℃、每30g湿菌体加入热水100mL、硫酸浓度0．25mol／L、搅拌转速160r／min,此时SAM的抽提率可达91．5％。与其他方法相比,该方法耗时少、仪器简单、抽提液中S-腺苷-L-甲硫氨酸质量浓度高、经济且无污染。     

酿酒酵母超氧物歧化酶(SOD)基因的克隆和表达

通过PCR扩增技术从酿酒酵母中得到了Cu,zn—SOD的结构基因,此基因被亚克隆到大肠杆菌质粒载体pT7—7．得到重组质粒pT7－7：SOD。利用EcoRI和Pstl酶切pT7-7::SOD质粒．经琼脂糖凝腔电泳,DEAE-滤膜回收Cu。zn—SOD结构基因片段,将其亚克隆到M13中．并转化大肠杆菌,得到了重组质粒M13-::t SOD,酶切和纯化后的SOD基因,定向克隆到酵母质粒载体pHz－8的smal和EcoRI位点上,构建成重组质粒pHZ－8－l。经转化酵母受体菌ZH-l和DP—l后得到了转化子．来自于ZH—l的转化子在非选择性条件下培养40世代后仍有95％以上细胞保留重组质粒。而来自于DP-1的转化子很不稳定。经蛋白提取、聚丙烯酰胺凝胶电泳和酶活性测定结果表明,来自于zH－1转化子中SOD的表达量约为细胞可溶性蛋白的15％．并具有生物活性。     

Production of Extracellular lipases by Saccharomyces cerevisiae

Seven strains of Saccharomyces cerevisiae all produced lipase when grown in shake flask culture. The best strain, DSM 1848, produced 4.0U of lipase in the medium containing olive oil and yeast extract. Production of the lipase was growth-associated.