转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

将人工合成的树干毕赤酵母（Pichia stipitis）的木糖还原酶基因XYL1插入酿酒酵母（Saccharomyces cerevisiae）表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达（13.68±2.37）g/L、（12.09±1.45）g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量（1.08±0.37）g/L（P＜0.05）,说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力（P＜0.05）。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。     

Cloning and Expression of AROIO Gene in Saccharomyces cerevisiae and It＇ s Influence on 3- （methylthio） -1-propanol Biosynthetic Metabolism

研究了脱羧酶ARO10基因克隆与过量表达对酿酒酵母INVSc1 3-甲硫基丙醇合成途径的代谢流量影响。将脱羧酶基因ARO10与穿梭质粒pYES2连接,构建其酿酒酵母表达质粒（载体）pYES2-ARO10,LiAc/SSD-NA/PEG方法转化酿酒酵母菌株INVSc1中进行表达,验证ARO10基因过量表达对发酵产物3-甲硫基丙醇影响。结果表明,构建的酿酒酵母转化子SC10-1发酵120 h时,3-甲硫基丙醇生成量达到0.90 g/L,与未导入脱羧酶ARO10基因的对照菌株相比,3-甲硫基丙醇产量提高55.2%。因此,S.cerevisiae s288c中脱羧酶（EC 4.1.1.72）是3-甲硫基丙醇生物合成途径的关键限速酶,其增强脱羧酶基因ARO10的克隆及基因表达,有利于提高3-甲硫基丙醇的合成代谢流量。     

PHA聚合酶基因phaC的克隆及其在酿酒酵母中的表达

对聚羟基脂肪酸酯(PHA)聚合酶基因phaC进行克隆,并将其与穿梭质粒pYES2连接.构建了酿酒酵母表达质粒pYES2-phaC.测序验证后用LiAc/SSDNA/PEG方法将质粒转化至Saccharomyces cerevisiae INVSCI中,经SC-URA培养基筛选得到阳性转化子.由2%半乳糖培养基诱导表达后,提取细胞粗蛋白测定PHA聚合酶酶活力,结果表明,表达phaC基因的重组菌酶活力为2.45 U/mg,证明phaC基因在在cerevisiae INVSCI中得到表达.     

纳豆激酶基因在酿酒酵母中的表达

目的:从纳豆芽孢杆菌基因组DNA中扩增纳豆激酶基因,以实现该基因在酿酒酵母中的表达.方法:通过PCR方法扩增纳豆激酶基因,利用DNA重组技术构建重组质粒pYES2-NK,转化酿酒酵母H158感受态细胞;经β-半乳糖诱导表达后,用纤维蛋白平板法检测纤溶酶活性.结果:克隆到大小为1192bp的纳豆激酶基因,编码397个氨基酸;活性检测表明酿酒酵母发酵液的上清液具有纤溶酶活性.结论:纳豆激酶基因在重组酿酒酵母中实现了分泌表达.     

甜蛋白monellin基因在酿酒酵母中的分泌表达

人工合成的单链甜蛋白monellin基因与经改造后的基因分别克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYES2.0中,得到表达载体pYESM及pYESMT。在表达载体pYESMT中,monellin基因的上游连接酿酒酵母的α-信号肽序列,得到分泌表达载体pYESMTA。分别将3个重组质粒转化酿酒酵母菌株INVsc1中进行表达。具有突变monellin基因的菌株INVMT/pYESMT的monellin蛋白表达量明显高于含monellin基因的菌株。而含有pYESMTA的菌株可将monellin基因分泌到胞外,表明α-信号肽序列能很好地将酿酒酵母中的重组monellin蛋白引导到胞外,完成分泌表达,且表达产物具有生物活性。     

ARO10基因在Saccharomyces cerevisiae中的克隆表达及其对3-甲硫基丙醇合成代谢的影响

研究了脱羧酶ARO10基因克隆与过量表达对酿酒酵母INVSc1 3-甲硫基丙醇合成途径的代谢流量影响。将脱羧酶基因ARO10与穿梭质粒pYES2连接,构建其酿酒酵母表达质粒(载体)pYES2-ARO10,LiAc/SSD-NA/PEG方法转化酿酒酵母菌株INVSc1中进行表达,验证ARO10基因过量表达对发酵产物3-甲硫基丙醇影响。结果表明,构建的酿酒酵母转化子SC10-1发酵120 h时,3-甲硫基丙醇生成量达到0.90 g/L,与未导入脱羧酶ARO10基因的对照菌株相比,3-甲硫基丙醇产量提高55.2%。因此,S.cerevisiae s288c中脱羧酶(EC 4.1.1.72)是3-甲硫基丙醇生物合成途径的关键限速酶,其增强脱羧酶基因ARO10的克隆及基因表达,有利于提高3-甲硫基丙醇的合成代谢流量。     

高效表达木糖醇脱氢酶基因酿酒酵母的构建及木酮糖发酵的初步研究

通过RT-PCR方法克隆得到Candida tropicalis木糖醇脱氢酶基因xyl2,将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下,构建表达质粒pYES2-xyl2;同时用从Pichia pastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因,构建含组成型启动子GAP基因的表达质粒pYES2-GAP-xyl2;通过电转化法将其依次转入酿酒酵母S.cerevisiae INVSc1,山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养,筛选出1株耐木糖醇浓度为20%的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15%的重组菌株YDX2。酶活测定表明,重组菌株ZCX4比酶活0.621 U/mg(蛋白),是YDX2比酶活的2.29倍。摇瓶发酵结果显示,重组菌株ZCX4木糖醇消耗76.46 g/L,木糖醇消耗率为76.46%,是重组菌株YDX2木糖醇消耗率的1.63倍,说明木糖醇脱氢酶实现了高效表达。     

瑞氏木霉纤维素酶基因在酿酒酵母中的表达研究

从瑞氏木霉（Trichoderma reesei）cDNA文库中克隆到外切葡聚糖纤维二糖水解酶Ⅰ（CBHI）eDNA基因，将该基因分别连接到酿酒酵母（Saccharomyces cerevisiae）表达载体pAJ401和pVT100_U上，构建了重组质粒pAJ401-cbhl和pVTIOO_U-cbhl。分别电转化2个重组质粒转移至酿酒酵母H158中，得到重组酵母HPC和HAC，实现了CBHI的分泌型表达。再将质粒pAJ401-cbhl转入已含有瑞氏木霉内切葡聚糖酶Ⅰ基因egl的重组酵母Hlm中，构建了同时表达cbhl和egl的重组酵母菌株HMEPC。比较HMEPC和Hlm对纤维素底物的降解，前者滤纸酶活比后者提高14．3％，表明CBHI与EGI对滤纸降解有协同作用。     

人α-防御素5前体蛋白在毕赤酵母中分泌表达的研究

为了探索人α-防御素5(HD5)前体蛋白基因在酵母中高效分泌表达的可行性,将其对应基因克隆到载体pPIC9K,得到重组载体pPIC9K-preproHD5,经SacI线性化后,转入Pichiapastoris GS115,得到重组毕赤酵母菌GS115/pPIC9K-preproHD5。应用PCR技术筛选高拷贝转化株,用BMGY培养GS115/pPIC9K-HD5至OD600为2～6时,用BMMY诱导培养120h,离心取上清液,用Tricien-SDS-PAGE和蛋白定量试剂盒分析人α-防御素5前体蛋白的表达量。琼脂扩散法检测人α-防御素5前体蛋白的抑菌活性,发现其对金黄色葡萄球菌(ATCC6538)和大肠杆菌(ATCC10231)2种标准菌株具明显的抑菌活性。人α-防御素5前体基因可在毕赤酵母实现分泌型表达,该策略可能成为防御素工业化开发的有效途径。     

甜叶菊糖基转移酶UGT76G1的克隆表达及其性质研究

目的:利用甜叶菊糖基转移酶UGT76G1特异性催化甜菊糖中含量高并且具有较强后苦味的甜菊甙生成高甜度的莱鲍迪甙A。方法:将合成的UGT76G1编码基因插入pYES2载体的EcoRⅠ和XhoⅠ酶切位点之间,成功构建了pYES2-UGT重组质粒。重组质粒导入表达宿主酿酒酵母YPH499中,利用2%半乳糖对重组菌进行诱导表达。结果:确定了最佳诱导时机为菌体培养后48h,诱导表达时间为12h。并对重组酶粗酶液性质进行了初步研究,确定其最适反应pH为8.0,最适反应温度为40℃,最佳反应时间为36h。结论:为建立经济高效的生物催化法对甜菊糖口味改质奠定了基础。     

西藏沙棘酵母菌的分离鉴定及其产香特性分析

以西藏隆子县野生沙棘为原材料分离酵母菌,经分离纯化后得到4 株具有良好发酵能力的酵母菌,经形态观察和26S rDNA测序,4 株酵母均为酿酒酵母菌（Saccharomyces cerevisiae）。对照商用酿酒酵母（ATCC 9763）进行生长特性、耐受性和发酵特性分析。结果表明,菌株Nysj-7耐乙醇能力（体积分数18%）高于菌株Nysj-4、Nysj-9和菌株ATCC 9763,与菌株Nysj-1相同,且耐SO2能力明显高于其他菌株;菌株Nysj-4、Nysj-7、Nysj-9和菌株ATCC 9763的耐葡萄糖质量浓度相同,均为500 g/L,高于Nysj-1;4 株分离酵母菌耐最低pH值相同,均为2.5;菌株Nysj-7、Nysj-1和Nysj-4产乙醇能力与商用酵母菌ATCC 9763相当,强于菌株Nysj-9;菌株Nysj-7产酯能力强于其他菌株,产硫化氢能力低于其他菌株,产β-葡萄糖苷酶能力高于菌株ATCC 9763,与菌株Nysj-1和Nysj-9差异不显著,但低于菌株Nysj-4;挥发性物质检测结果表明,菌株Nysj-7发酵的沙棘果酒中挥发性物质524 种,与ATCC 9763相比,差异代谢物95 种,其中上调表达挥发性物质69 种,下调表达26 种,且上调表达的香气物质相对含量显著高于下调表达的香气物质相对含量;主成分分析结果表明,菌株Nysj-7与ATCC 9763相比,其挥发性物质在表达量上存在明显差异。综上所述,Nysj-7菌株具有良好的发酵特性和耐受性,可用于果酒酿造。     

Human pancreatic glucokinase (GlkB) complements the glucose signalling defect of Saccharomyces cerevisiae hxk2 mutants

Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae. The recombinant protein showed similar enzyme kinetics to those described for the original enzyme. When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant. It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart.     

重组酿酒酵母合成白藜芦醇的研究

为了获得一种酵母快速合成白藜芦醇的体系,本研究分别从虎杖和烟草中克隆获得白藜芦醇合成途径的关键酶基因:芪合酶基因STS和4-香豆酰辅酶A连接酶基因4CL,引入3个连续重复的GSG作为连接肽,采用Overlap PCR扩增技术构建了融合基因4CL-(GSG)3-STS,进一步获得重组表达载体p ESC-TRP-4CL-(GSG)3-STS后转化至酿酒酵母中,之后利用HPLC分析检测重组酿酒酵母代谢产物,最后对重组菌合成白藜芦醇的诱导时间、底物添加浓度和添加方式进行了优化研究。结果表明:所构建的重组酿酒酵母菌体生长48 h后进行诱导表达,同时添加浓度为6 mg/L底物4-香豆酸,并且每隔2 h添加一次,8 h后白藜芦醇产量即可达7.01 mg/L。利用本体系合成白藜芦醇具有操作简单、生产周期短的特点,为进一步实现微生物大规模生产白藜芦醇提供了基础。     

酿酒酵母表面展示β-淀粉酶及其酶学性质研究

通过PCR技术扩增来源于大麦的β-淀粉酶基因,将其与酿酒酵母细胞壁蛋白α凝集素基因在读框内融合,构建得到表面展示载体pBA-AG,进一步将该重组质粒通过遗传转化,整合到酿酒酵母W303-1A的染色体中,获得了β-淀粉酶经过α凝集素锚定信号结合到细胞壁上的重组酵母。重组酵母表面展示的β-淀粉酶活力为131U/g干细胞。对展示的β-淀粉酶酶学性质研究表明,其最适反应温度为50℃,最适作用pH为5.0,与游离酶相比,其温度稳定性和pH稳定性均得到提高。本研究利用α凝集素系统首次将β-淀粉酶成功展示在酿酒酵母表面,为以酿酒酵母为基础的全细胞催化剂研究与应用打下了一定基础。     

Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses

The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.     

Bioethanol production from xylose by recombinant Saccharomyces cerevisiae expressing xylose reductase, NADP(+)-dependent xylitol dehydrogenase, and xylulokinase

We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.     

Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion.     

超声对酵母细胞膜通透性的影响

用不同参数的变幅杆浸入式超声波作用酵母细胞,测定细胞内核酸、蛋白质及FDP(1,6-二磷酸果糖)3种物质渗出率,再用透射电镜观察细胞形态。探讨超声场对酵母细胞膜通透性影响的最佳参数及机理。结果表示,超声场的最佳参数是:电功率500W、脉冲时间3s、间隙时间4s、脉冲总时间共225s,影响酵母细胞膜通透性变化的主要机制是空化作用使细胞膜在一定程度上损伤产生穿孔效应,细胞膜的通透性提高。