西方许旺酵母α-淀粉酶的基因克隆及其在啤酒酵母中的高效表达与分泌

以E .coli/yeast穿梭质粒YCEp1为载体构建许旺酵母部分基因组文库 ,在大肠杆菌中扩增后提取混合质粒DNA ,经电转化非缺陷标志啤酒酵母AS .2 1 36 4 ,在YPDS平板 ( 1 %葡萄糖和 1 %可溶性淀粉 )上用淀粉水解活力筛选含水解淀粉能力的阳性转化子 .从阳性转化子中分离融合质粒证实含 5 0kb的插入片段 .用α 淀粉酶基因两端序列设计的引物PCR扩增及扩增片段序列分析证实该片段中含有α 淀粉酶全部编码序列 .该片段能在啤酒酵母中表达α 淀粉酶 ,说明该片段带有α 淀粉酶基因 5′ 上游序列和 3′ 下游序列 .该转化子在YPDS平板 30℃培养 48h形成清晰可见的水解淀粉圈 .发酵液 (YPDS) 48℃可获得 740～ 780mU/mL产酶活力和高的生物量 .PAGE证实发酵液有α 淀粉酶蛋白带 ,占胞外总蛋白含量的 1 2 %以上 .说明在载体YCEp1上许旺酵母α 淀粉酶基因自身启动子 ( promoter)和终止子(terminator)在啤酒酵母AS .2 .1 36 4中同样被识别且高效表达 .有工业应用前景的水解淀粉酵母菌株构建成功 .     

枯草杆菌α-淀粉酶基因克隆及其在毕赤酵母中表达的初步研究

以B.subtilis XL-15基因组为模板,运用PCR法成功克隆了α-淀粉酶基因,其开放式阅读框(ORF)为1980bp,编码659个氨基酸残基。分别将该基因转入大肠杆菌BL21(DE3)和毕赤酵母GS115中,进行诱导表达。结果表明,大肠杆菌破碎上清液中未检出酶活,SDS-PAGE电泳分析显示表达产物均以无活性包涵体存在;而毕赤酵母在α-Factor及AOX1基因启动子和终止信号的调控下,经高密度培养,表达产物分泌至胞外,发酵液酶活力为4.3U/ml,实现了B.subtilis α-淀粉酶基因的分泌表达。     

α-淀粉酶在短短芽孢杆菌中的分泌表达

应用PCR技术从枯草杆菌168中分离出α－淀粉酶基因。将其引入分泌表达载体pBKE50后,用Tris-PEG法转入短短芽孢杆菌50中,发现α－淀粉酶以活性形式被分泌表达。酶测活分析表明α－淀粉酶活性约为出发菌枯草杆菌168的1.7倍。     

黑曲霉α-葡萄糖苷酶基因的克隆及其在毕赤酵母中的表达

【目的】以毕赤酵母（Pichia pastoris）KM71为宿主菌表达黑曲霉（Aspergillus niger）SG136 α-葡萄糖苷酶（α-glucosidase）。【方法】以实验室保藏的A. niger SG136总DNA为模板,根据NCBI 数据库中A. niger CBS 513.88 α-葡萄糖苷酶的cDNA序列（aglu）设计引物,通过PCR和重叠延伸PCR（overlap-PCR）方法,扩增得到aglu,将其克隆到载体pMD18-T simple vector,测序结果表明,aglu编码960个氨基酸。与A. niger CBS 513.88 α-葡萄糖苷酶相比仅有一个氨基酸的差异。将得到的aglu亚克隆到质粒pPIC9K,构建表达载体pPIC9K-aglu,经Bgl Ⅱ线性化后电转化P. pastoris KM71,经过MD、YPD/G418平板筛选表型,PCR方法验证,获得分泌表达重组P. pastoris KM71/pPIC9K-aglu。摇瓶培养中通过添加终浓度为1 %的甲醇诱导α-葡萄糖苷酶的分泌。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为98 kDa和33 kDa,阴性对照中没有出现此条带,非变性电泳检验为一条带。制备的粗酶液的酶学性质表明,转苷反应最适pH为5,最适温度为55 ℃。在最适pH和温度下,反应24 h时低聚异麦芽糖的总含量达到最大为26.0 %。【结论】黑曲霉α-葡萄糖苷酶在P. pastoris中获得可溶性表达,并证明有一定的转糖苷活性。     

黑曲霉酸性α-淀粉酶基因和糖化酶基因对工业酒精酵母的整合及其共表达

用PCR合成的黑曲霉 (Aspergillusniger)酸性α 淀粉酶 (Acidα amylase)的cDNA ,构建了由酵母醇脱氢酶(ADH1 )启动子和终止子引导表达 ,酸性α 淀粉酶自身信号肽序列引导分泌的表达元件 ,与黑曲霉糖化酶cDNA的表达元件同时插入由酵母rDNA序列引导同源整合的酵母YIp型表达载体pWHY中 ,构成双基因表达分泌质粒pWAG。采用pWAG与酵母YEp型G4 1 8抗性表达质粒的共转化 ,将两个基因表达元件整合到酒精生产酵母菌株AS2 346的染色体rDNA序列中 ,获得同时表达胞外酸性α 淀粉酶和糖化酶的双功能酒精酵母工程菌     

合成耐高温α-淀粉酶PFA在巴斯德毕赤酵母中的分泌表达

PFA是来源于Pyrococcus furious的一种耐高温α-淀粉酶,为了使PFA能够在巴斯德毕赤酵母中高效表达,根据巴斯德毕赤酵母密码子的偏好性对PFA的基因序列进行密码子优化,人工合成耐高温淀粉酶PFA基因pfa,并连接到巴斯德毕赤酵母中表达载体pPIC9K上,得到重组质粒pPIC9K-pfa。重组质粒线性化后转化到巴斯德毕赤酵母菌株GS115中,重组菌株在摇瓶中用甲醇诱导表达,分泌表达酶活最高为220U／L。     

超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达

用PCR方法扩增来源于极端嗜热厌氧古菌Pyrococcus furiosus中的超耐热酸性α-淀粉酶的结构基因,将该结构基因引入载体pPIC9K中,将重组质粒pPIC9K-Amy转化大肠杆菌DH5α细胞,测序结果表明,克隆到的α-淀粉酶结构基因为1305bp,其编码的成熟肽为435个氨基酸。将正确构建的重组质粒转化毕赤酵母GS115细胞,得到酵母工程菌株。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,超耐热酸性α-淀粉酶在甲醇酵母中大量表达并分泌到胞外,该酶的表达受甲醇的严格调控和诱导,随着诱导培养时间的增加,在培养基上清液中的单位体积酶活力相应上升,在诱导培养7d后酶活力达到最大值。该酶最适反应温度为90~100℃,最适反应pH值为4.5~5.5。该酶具有非常好的温度稳定性,在100℃条件下热处理5h,仍具有60%以上的酶活力。该酶的这些优点使其非常适于在工业生产上应用。     

猪IFNα基因在毕赤酵母中的高效分泌表达

巴斯德毕赤酵母载体质粒pPICZαA含有强启动子PAOX1和α-MF信号肽序列,构建猪IFNα基因的重组质粒pPICZαA-IFNα,并转入E.coli JM109中,得到转猪IFNα基因工程菌,经酶切鉴定克隆到载体pPICZαA上的外源基因即为猪IFNα基因。通过电击将经SacⅠ酶切后线性化的pPICZαA-IFNα质粒转化到巴斯德毕赤酵母KM71中。SDS-PAGE和Western blot鉴定表达产物的结果表明,分泌于胞外的猪IFNα蛋白分子量比猪IFNα理论值分子量稍大,估计是糖基化的原因。表达的蛋白可发生正确的抗原-抗体反应,表达量为 0.45 mg/mL。将蛋白表达上清经细胞毒性实验检测表达产物的抗病毒活性为2.1×104 IU/mL。Abstract: The porcine alpha interferon gene was inserted into the Pichia pastoris expression vector of pPICZαA which contains AOXⅠpromoter and α-factor signal sequence.The recombinant plasmid was transformed into host cell E.coli JM109 and then was extracted for analysis of restriction enzymes.It was confirmed that heterogeneous gene spliced into vector pPICZαA was IFNα gene. The recombinant plasmid of pPICZαA-IFNα was linearnized by SacⅠand transformed into KM71 by electroporation. SDS-PAGE and Western blot analysis showed that IFNα product was observed in the supernants with a little larger molecular weight size than the natural IFNα.The rIFN gene has the same antigenicity as natural one.The expressed rIFN accumulated up to about 0.45mg/mL.The cytokine activity of the supernants was vertified by WISH/VSV system,which is about 2.1×104IU/mL.     

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。     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Amylase production by a Schwanniomyces occidentalis mutant in chemostat culture

The crystalline cell surface layer (S-layer) from Bacillus stearothermophilis PV72 was used as a matrix for reversible immobilization of -d-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2-dipyridyldisulphide, 550 g -d-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one -d-galactosidase molecule [relative molecular mass (M_r), 116000] per two S-layer subunits (M_r, 130 000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies -d-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.Correspondence to: M. Sára     

Cloning and Expression of a Schwanniomyces occidentalis α-Amylase Gene in Saccharomyces cerevisiae

An α-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of α-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for α-amylase synthesis. The concentration of α-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted α-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Secretion of invertase from Schwanniomyces occidentalis

Invertase synthesis in Schwanniomyces occidentalis is regulated by catabolite repression and is derepressed by raffinose and low concentrations of glucose. Efficiency of a carbon source in derepression of invertase is dependent upon the type of culture medium: either raffinose in a rich medium or a low concentration of glucose in a yeast minimal medium. The kinetics of derepression can be modulated by changing the carbon source. When cells are grown in a rich medium with 0.5% raffinose as the sole carbon source, Schwanniomyces occidentalis secretes 80 times more invertase than Saccharomyces cerevisiae grown in the same conditions. About 50% of the total amount of invertase produced by Schwanniomyces occidentalis is secreted in the extracellular medium in contrast to Saccharomyces cerevisiae where only 6 to 15% of the protein is secreted in the medium.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Transformation system for Schwanniomyces occidentalis

Spheroplasts of Schwanniomyces occidentalis were used for a transformation system. A putative leu2 mutant of S. occidentalis was complemented with the LEU2 gene (YEp13) from Saccharomyces cerevisiae. The transformation efficiency was 10 3 transformants/mg DNA. Although low stability was obtained, YEp13 could be recovered from transformants and kept the same size and restriction enzyme cutting sites like the original one. The replicon of 2 mm plasmid is responsible for the replication of YEp13 in S. occidentalis.     

Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K(m)) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.     

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis.

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs (J. D. Beggs, Nature [London] 275:104-108, 1978). Intact plasmids were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.