glyA基因的克隆及其在毕赤酵母中的表达

根据已知的序列设计引物,以大肠杆菌XL10-Gold总DNA为模板进行梯度PCR,并进行DNA序列测定,其序列与已经报道的glyA基因完全一致。将其克隆到毕赤酵母分泌型表达载体pHBM905C上,获得表达质粒pHBM1001.该质粒转化毕赤酵母GS115所得重组子经PCR验证后成功进行了诱导表达,并初步测定了酶活力。     

脂肪酶1在毕赤酵母中的高效表达

根据褶皱假丝酵母脂肪酶CRL中LIP1的成熟多肽序列 ,人工合成了由毕赤酵母 (Pichiapastoris)中偏爱密码子组成的lip1基因序列。该基因以N端融合的方式分别正确插入毕赤酵母诱导型表达载体pPICZαA与组成型表达载体pGAPZαA中。通过电激将线性化的上述两种重组质粒分别转化毕赤酵母SMD116 8H细胞 ,筛选获得两株分别具有诱导型表达和组成型表达生物活性LIP1能力的高产菌株。其中 ,组成型表达菌株CHT II换液后 4 8h上清液中含脂肪酶活力为 2 .0 0× 10 5u/L ,表达产物在pH 4～ 8与温度 30～ 5 0℃范围内具有较高的脂肪酶活性 ;高密度发酵条件下 ,发酵 72h ,上清液中含脂肪酶活力可达 1.395× 10 6u/L ,表明构建的重组菌株具有更大的工业化生产优势。     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶（PEL）的cDNA克隆到酵母整合型质粒pPIC3.5K,电转化His4缺陷型巴斯德毕赤酵母(Pichia pastoris)GS115,通过橄榄油MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDSPAGE分析、橄榄油检验板鉴定,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中,分子量约28kD,与扩展青霉碱性脂肪酶大小一致,占分泌蛋白的95%。橄榄油检验板检验表明该表达蛋白可分解橄榄油,通过优化该表达菌的发酵条件,以橄榄油为底物进行酶活测定,其发酵液酶活可达260 u/mL。     

二步法黑曲霉脂肪酶基因lipA的全基因合成及其在毕赤酵母中的高效表达

黑曲霉脂肪酶是重要的工业用酶,在食品、制药等领域具有广泛的用途。获得黑曲霉脂肪酶高效表达的基因工程菌是该脂肪酶规模化应用的前提。通过全基因合成对目的基因进行分子改造和人工组建是实现基因高效表达和体外分子进化的有效手段。本研究主要针对一步法长片段基因合成中复杂结构的非特异性配对和过多的PCR引入碱基错配等问题,采用二步法(组装PCR和酶切-酶连)合成了黑曲霉脂肪酶基因lipA。首先在DNA2.0和Gene2Oliga软件辅助下对lipA基因密码子及RNA二级结构进行优化并引入ClaI(237位)和PstI(475位)酶切位点;通过组装PCR分别合成lipA基因的各片段F1(237bp)、F2(238bp)和F3(422bp);通过该基因内的ClaI和PstI限制性酶切位点连接成完整的全长lipA基因。本方法有效地降低了长片段合成过程中寡核苷酸片段的非特异性配对、复杂的二级结构以及碱基突变对DNA合成的影响,提高了长片段合成的成功率。经密码子优化后的脂肪酶基因(lipA-syn)在毕赤酵母GS115中经诱导表达72h后,发酵液酶活达176.0U/mL,蛋白质含量为143.7mg/L,较出发基因分别提高了10.8倍...     

mdlA基因在毕赤酵母中的高效表达及表达产物性质研究

将编码甘油单-二酰酯脂肪酶(MDGL)的基因mdlA插入到分泌表达质粒pPIC9K中,通过电激将线性化的重组质粒整合到毕赤酵母(Pichia pastoris)GS115中,筛选出H is Mut 表型菌株,进一步用G418筛选获得高拷贝转化子,并用PCR方法鉴定。诱导培养后,SDS-PAGE表明MDGL在毕赤酵母中得到有效表达。表达产物在温度40℃,pH7.5具有最高活性,其发酵液酶活可达到325U/mL,以橄榄油为底物时没有检测到活性。表达产物与甘油三酰酯脂肪酶共同作用时产生的脂肪酸量比甘油三酰酯脂肪酶单独作用提高了93.5%。     

纳豆激酶基因的克隆及其在毕赤酵母中的表达

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。     

狐狸生长激素基因的克隆及其在毕赤酵母中的表达

为制备重组狐狸生长激素(fGH),采用RT-PCR方法,从银狐垂体中扩增fGHcDNA基因,利用SnaBI和NotI位点将fGH基因插入到酵母分泌型表达载体pPIC9K中α-因子信号肽的下游,构建成fGH基因的酵母分泌型表达载体pPIC9K/fGH,载体经SalI酶切线性化后,通过电转移将线性化的pPIC9K/fGH转化到组氨酸缺陷型酵母宿主菌GS115中。然后利用不含氨基酸的以葡萄糖为碳源的培养基(MD)和以甲醇为碳源的培养基(MM)筛选出组氨酸His+型和甲醇利用正型(Mut+)酵母重组体,再经G418加压筛选出高拷贝fGH基因的重组酵母,经摇瓶发酵培养和甲醇诱导使fGH进行分泌表达。结果表明本实验扩增的fGH基因序列与GenBank发表的序列基本一致,发酵液经SDS-PAGE和Western blotting检测证明构建的重组酵母能够分泌表达fGH,表达的fGH占发酵液总蛋白的34%,表达量达119mg/L发酵液。     

枯草芽孢杆菌(Bacillus subtilis)碱性脂肪酶基因在毕赤酵母中的表达

将来自枯草芽孢杆菌的碱性脂肪酶基因经密码子优化,全基因合成后克隆到pPICZαA载体,构建了pPICZαA-bsl分泌型重组质粒,该重组质粒经限制性内切酶PmeI线性化后使用LiCl法转化到毕赤酵母X-33,经过筛选获得分泌表达碱性脂肪酶的重组毕赤酵母X-33/pPICZαA-bsl。摇瓶发酵液上清酶活最高可达4.78 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为40-60℃,最适pH9.0,且具有高度耐碱的特性。该重组脂肪酶对旧新闻纸具备较明显的脱墨能力。     

The TGL2 Gene of Saccharomyces cerevisiae Encodes an Active Acylglycerol Lipase Located in the Mitochondria

The Saccharomyces cerevisiae Tgl2 protein shows sequence homology to Pseudomonas triacylglycerol (TAG) lipases, but its role in the yeast lipid metabolism is not known. Using hemagglutinin-tagged Tgl2p purified from yeast, we report that this protein carries a significant lipolytic activity toward long-chain TAG. Importantly, mutant hemagglutinin-Tgl2p^S144A, which contains alanine 144 in place of serine 144 in the lipase consensus sequence (G/A)XSXG exhibits no such activity. Although cellular TAG hydrolysis is reduced in the tgl2 deletion mutant, overproduction of Tgl2p in this mutant leads to an increase in TAG degradation in the presence of fatty acid synthesis inhibitor cerulenin, but that of Tgl2p^S144A does not. This result demonstrates the lipolytic function of Tgl2p in yeast. Although other yeast TAG lipases are localized to lipid particles, Tgl2p is enriched in the mitochondria. The mitochondrial fraction purified from the TGL2-overexpressing yeast shows a strong lipolytic activity, which was absent in the tgl2 deletion mutant. Therefore, we conclude that Tgl2p is a functional lipase of the yeast mitochondria. By analyzing phenotypic effects of TGL2-deficient yeast, we also find that lipolysis-competent Tgl2p is required for the viability of cells treated with antimitotic drug. The addition of oleic acid, the product of Tgl2p-catalyzed lipolysis, fully complements the antimitotic drug sensitivity of the tgl2 null mutation. Thus, we propose that the mitochondrial Tgl2p-dependent lipolysis is crucial for the survival of cells under antimitotic drug treatment.     

米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。     

mMR-1基因的克隆和在毕赤酵母中分泌表达

hMR-1为本实验室首次克隆发现的一个人类新基因 ,是一种和三种肌肉收缩蛋白及多种细胞信号蛋白具有相互作用的膜蛋白。经与鼠基因组数据库进行同源分析后设计合成引物 ,利用RT-PCR技术从小鼠C57BL 6J脾脏T淋巴细胞总RNA中反转录得到鼠源MR-1基因 (mMR-1) ,提交GenBank ,收录号 (AY2 99972 )。序列分析证明其与人源MR-1基因 (hMR-1)同源性为 90.1%。构建表达载体pPIC9 mMR-1电转化PichiapastorisGS115 ,筛选得到整合分泌表达mMR-1蛋白的重组酵母菌株。表达目的蛋白分子量 25kD ,诱导 5d时产量达到 50mg/L ,通过Westernblot验证了表达产物的正确性。本研究为进一步研究新基因MR-1的生物学功能奠定了基础。     

长梗木霉内切葡聚糖酶I基因的克隆及其在毕赤酵母中的表达

一株纤维素酶高产菌株经ITS序列鉴定并命名为长梗木霉SSL (Trichoderma longibrachiatum, SSL)。利用RT-PCR的方法从该菌株中克隆出内切-1-4-β-D-葡聚糖酶I的基因 (eg1), 该基因全长1386 bp, 编码461个氨基酸。序列分析表明：该基因序列与T. longibrachiatum egl1基因具有90％以上的同源性。将该基因的成熟肽编码序列插入到Pichia pastoris表达载体ppic9k中, 构建重组表达质粒ppic9k-eg1, 转化P. pa     

Cloning of the Pichia anomala SEC61Gene and Its Expression in a Saccharomyces cerevisiae sec61 Mutant

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene. Received: 24 May 2002 / Accepted: 10 July 2002     

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.