重组巴斯德毕赤酵母高密度发酵表达植酸酶

对巴斯德毕赤酵母的高密度发酵条件进行了试验,并根据摇瓶发酵的优化结果进行了补料方式的研究。在摇瓶发酵时,最佳种龄为16h,接种量为3％,甲醇的诱导浓度为15g／L,生长阶段最适pH为5．0,诱导阶段最适pH为5．5。在间歇补料、恒速补料、变速补料三种补料方式中以变速流加最优。     

重组毕赤酵母表达工程植酸酶发酵过渡相参数相关分析

微生物发酵是一个涉及不同尺度的互相关联的复杂生物系统的过程 ,将重组毕赤酵母表达工程植酸酶过渡相的在线和离线参数进行了相关分析研究。通过对发酵过程的在线细胞代谢生理参数 (OUR)和环境参数 (DO)的变化进行相关分析表明 :甘油和葡萄糖碳源对AOX合成的阻遏强度不同 ,葡萄糖的阻遏性明显强于甘油 ,相对于醇氧化酶启动子 ,葡萄糖为强阻遏性底物。根据甲醇代谢途径关键酶酶活性变化 ,推测出各代谢途径流量分布的变化 ,即甲醇诱导后糖酵解途径和三羧酸循环途径代谢流比例下降 ,而磷酸戊糖途径中代谢流通量上升 ,甲醇完全氧化代谢流成为主要代谢流 ,与过渡相在线参数pH、OUR(CER)和RQ等相关分析的甲醇代谢途径的变化结果一致。此外 ,建立了生产过程在线控制与分析的标准 :当OUR和CER逐渐增大 ,则可判断甲醇已被利用和启动子已被甲醇成功诱导 ,即工程植酸酶开始启动表达.     

卡氏德巴利酵母植酸酶在毕赤酵母中的异源表达及优化

【背景】植酸是一种能螯合金属离子和蛋白质的有机磷类化合物,广泛存在于植物组织中,影响动物对营养元素的吸收。在饲料中加入植酸酶可有效降解植酸。【目的】构建毕赤酵母异源表达卡氏德巴利酵母(Debaryomyces castellii,D. castellii)植酸酶的菌株,促进卡氏德巴利酵母植酸酶的研究及工业应用。【方法】将卡氏德巴利酵母植酸酶基因进行密码子优化后转入毕赤酵母GS115中,通过筛选多拷贝、敲除蛋白酶、过表达分子伴侣及转运蛋白的方法获取优势菌株。【结果】所得重组菌株GS115/DCphy(ΔPep4)(BFR2)的产酶酶活是低拷贝菌株的7倍。【结论】研究结果为卡氏德巴利酵母植酸酶的异源表达及潜在工业应用提供了一定的指导。     

米曲霉植酸酶基因的克隆及其在毕赤酵母中的表达

根据已发表的植酸酶基因phyA序列(GI：4815609和GI：22901877)设计引物,以米曲霉(Asperillus oryae Cohn,编号：ACCC30155)的总DNA为模板,利用PCR扩增得到目的片段。将此片段克隆到表达载体pPIC9K的EcoRI酶切位点构建重组质粒,通过电转化方式将重组质粒转化毕赤酵母(Pichia pastoris)。检测结果表明,植酸酶基因在毕赤酵母中得到了表达。     

植酸酶基因的多点突变及在毕赤酵母中的高效表达

根据毕赤酵母基因的密码子选择偏爱性,不改变其编码氨基酸序列,对来源于黑曲霉N25植酸酶phyA基因,进行了突变,构建了含有正确突变的酵母表达载体pPIC9k-phyAm-4,电击转化毕赤酵母,获得优化了密码子的重组酵母转化子。经PCR鉴定表明,植酸酶基因已整合到酵母基因组中; 表达产物的SDS-PAGE分析表明,酶蛋白分子大小为70.15KD。Southern blotting结果表明,phyA基因整合到酵母染色体DNA中;转化子酶活测定结果表明,经密码子优化的重组酵母PP-NPm-4-2酶活可达136900U/ml,比Arg没有优化的PP-NPm-8 (47600 Uoml-1)酶活高约2.8倍。     

重组巴氏毕赤酵母高密度发酵表达rHSA

对基因工程菌Pichiapastoris的摇瓶发酵条件进行了试验 ,并根据摇瓶发酵的优化结果进行了补料分批高密度发酵。在摇瓶发酵时 ,甲醇诱导基因工程菌P .pastoris表达重组人血清白蛋白的发酵周期为 96h ;甲醇的最佳诱导浓度为 1 0g L ;发酵pH范围为 5 72～ 6 5 9;在摇瓶培养时 ,随着接种量的增加 ,虽然目的蛋白表达量缓慢增加 ,但单位细胞光密度的蛋白产率却明显下降 ,符合y =1 2 941x- 0 50 59方程 (线性相关系数r=0 9789) ,其限制性因子很可能为溶氧。在分批发酵 ,接种量为 1 0 %且种子细胞光密度 (OD60 0 )为 2 0左右时 ,细胞生长的延迟期为 2 1 1h左右 ,细胞生长光密度与培养时间的关系模型为 :y =0 7841e0 .2 3 19t(线性相关系数r=0 .993 6 ) ;在补料发酵时细胞干重浓度可达到 1 1 5g L— 1 6 0g L ,在 1 2 0h重组人血清白蛋白表达量最大达到 3 6g L。     

高效表达具有生物学活性的植酸酶的毕赤酵母

对来源于Aspergillusniger 96 3的植酸酶基因 phyA2进行了改造 ,包括去掉基因中的内含子和信号肽编码序列 ,在不改变所编码氨基酸的情况下 ,定点突变优化了对此基因在酵母中高效表达起关键作用的Arg密码子 .经改造的植酸酶基因按正确的阅读框架融合到毕赤酵母 (Pichiapastoris)高效表达载体 pPIC9上的α 因子信号肽编码序列 3′端 ,重组表达载体电击转化毕赤酵母得到重组转化子 ,经Southernblotting分析证实了植酸酶基因已整合到酵母基因组中 ,并确定了基因整合的拷贝数 .Northernblotting分析证明植酸酶基因能进行正常的转录 .SDS PAGE分析和表达产物的酶活性研究证明 ,植酸酶能有效分泌和高效表达 (Arg密码子优化后其表达量可达每毫升培养液 1 5 0 0 0U ,比Arg密码子没有优化的表达量高约 37倍 ,比原菌株A .niger 96 3的表达量高约 30 0 0倍 ) ,表达产物具有正常的生物学活性 .这是迄今为止我国饲料工业中 ,构建的第 1株具有实用价值和应用前景的生产饲料添加剂的基因工程菌株     

来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

黑曲霉N25植酸酶phyA基因在巴斯德毕赤酵母中高效表达的研究

从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植酸酶phyA基因全序列进行了比较。以此片段构建成功了pPIC9K-phyA载体(命名为pPNP-1),并转化毕赤巴斯德酵母。经G418抗性筛选、酶活性测定、Southern印迹和Western印迹,获得了高效表达的转化子PP-NP-1(23869.4u/ml)、PP-NP-2(20533.0u/ml)、PP-NP-3(35646.7u/ml),其酶活性分别是出发菌株的酶活(513.4u/ml)的46.46倍、39.99倍和69.46倍,且转化子具有很好的遗传稳定性。     

毕赤酵母中植酸酶和内切葡聚糖酶共表达菌株的构建及其高效表达(英文)

植酸酶和内切葡萄糖苷酶广泛应用于动物饲料添加剂中,能更有效地帮助饲料的利用,同时为动物的生命活动提供必需的重要物质。首先构建重组质粒pPICZα-EG,经过线性化后,电转化到含有植酸酶phyA基因的毕赤酵母感受态细胞中,构成含有植酸酶基因和内切葡萄糖苷酶基因的重组体菌株GS115-phyA-EG。经甲醇诱导后其活性分别达到出发菌株GS115-phyA和GS115-EG的39.4%和56.2%。酶学性质的分析显示,最适反应温度和最适反应的pH值分别为55℃和5.5,植酸酶和内切葡萄糖苷酶在温度为45℃~55℃,pH值为4.5~5.5时,酶活相对稳定,均能达到最高酶活的80%以上。结果表明这种在同一个系统种同时表达2种酶的方法能更好地节约时间和成本,使其在饲料工业的应用上有更广阔的前景。     

Recombinant Pichia pastoris overexpressing bioactive phytase

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High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.     

Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris . This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50–55 °C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.     

双碳源流加对重组毕赤酵母高效表达葡萄糖氧化酶的影响

葡萄糖氧化酶(GOD)是一种具有广泛应用前景的工业酶.为了实现葡萄糖氧化酶的高效生产,提高重组毕赤酵母生产GOD的产量和增强生产强度,对重组毕赤酵母诱导阶段的初始菌体浓度和甲醇浓度进行了优化.在此基础上,诱导期采用了双碳源(甘油、山梨醇和甘露醇)与甲醇混合流加的模式.研究发现,最佳诱导前初始菌体浓度和甲醇浓度分别为100 g/L和18 g/L,此时GOD产量为427.6 U/mL.在诱导阶段采用甘油、山梨醇和甘露醇与甲醇的混合添加均可以提高GOD产量,其中甘露醇与甲醇的混合流加效果最为显著.当甲醇与甘露醇混合流加的比例为20∶1(W/W)时,诱导156h GOD产量和生产强度分别可达711.3 U/mL和4.60 U/(mL·h),比甲醇单一流加策略结果分别提高了66.3％和67.9％.此外采用合适的甘露醇混合流加策略不但不会抑制AOX1启动子的表达,甚至有一定促进作用,AOX酶活性为8.8 U/g(对照为5.2 U/g).双碳源流加方式还能推广到毕赤酵母其他表型中,为该系统高效表达外源蛋白提供一种新策略.     

Production process for recombinant human angiostatin in Pichia pastoris

A pilot-scale production method of recombinant human angiostatin, a 38-kD fragment of plasminogen which has been reported to have antiangiogenic activity, has been successfully established by expressing the protein in the methylotrophic yeast Pichia pastoris. The secreted protein inhibited cultured endothelial cell proliferation in vitro and Lewis lung carcinoma growth in mice. The fermentation process was carried out using an on-line methanol controller, administering methanol to the growing culture and keeping its concentration under 2 g L⁻¹. The fermentation lasted 90 h, of which 70 h were growth on methanol. During growth on methanol the culture volume increased 64%, from 7 L to 11.5 L, producing 200 mg angiostatin and 5 kg of biomass. Journal of Industrial Microbiology & Biotechnology (2000) 24, 31–35. Received 12 May 1999/ Accepted in revised form 06 September 1999     

Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed andsynthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering theprotein sequence.The expression vector,pP9K-phyCs,was linearized and transformed in P.pastoris.Theyield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium(BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,respectively,improving over 90 folds compared with the wild-type isolate.Purified enzyme showed temperature optimumof 70℃ and pH optimum of 7.5.The enzyme activity retained 97% of the relative activity afterincubation at 80℃ for 5 min.Because of the heavy glycosylation the expressed phytase had a molecularsize of approximately 51 kDa.After deglycosylation by endoglycosylase H (EndoH_f),the enzyme had anapparent molecular size of 42 kDa.Its property and thermostability was affected by the glycosylation.     

发酵重组Pichia pastoris生产腺苷甲硫氨酸的研究

在5L发酵罐中对高产S腺苷甲硫氨酸的重组Pichia pastoris发酵进行了研究。重组菌在pH5．0生长,然后调为pH6．0积累腺苷甲硫氨酸,在30℃、溶氧5％及流加甲硫氨酸和尿素的条件下培养82h后,产量达4．3g／L。     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。