表达淀粉酶、糖化酶葡糖酸醋杆菌工程菌的构建

采用基因融合技术,将葡糖酸醋杆菌Gluconacetobacter hansenii ATCC23769分泌蛋白CMCax的信号肽序列分别与来源于枯草芽胞杆菌的淀粉酶基因、黑曲霉的糖化酶基因融合构建融合蛋白,连入能在G.hansenii ATCC23769自主复制的载体pbs-H1S中,电击转入G.hansenii ATCC23769,构建能内源表达淀粉酶、糖化酶,以及淀粉酶-糖化酶的葡糖酸醋杆菌。淀粉平板透明圈检测结果和DNS测酶活结果显示,构建的3种工程菌能成功表达并分泌淀粉酶和糖化酶。     

日本育成葡糖淀粉酶重组酵母菌

1986年在大阪召开的大阪发酵工程学年会上,三得利啤酒公司应用微生物研究所和酒类研究所发表了一项研究成果。用DNA组入染色体的方法选育成功稳定的基因重组酵母菌。将根霉的葡糖淀粉酶基因组入乙醇发酵能力接近于生产菌的酵母菌中,使其分泌产生葡糖淀粉酶,而无需加热处理(无蒸煮)就能由淀粉产生13％以上的乙醇。特别在用木薯淀粉为原料时分解率很高。转化为乙醇的产率与现行工业生产法相同。为使染色体中重组性能稳定,从小瓶到大容器重组体经数十代仍可稳定产生葡糖淀粉酶。 此重组体有可能在良好的通用发酵罐中实现工业化。该公司已实现用根霉的葡糖淀粉酶进行淀粉的无蒸煮乙醇生产,这种方法由淀粉产乙醇的能耗量约减少了三成。     

黑曲霉葡萄糖淀粉酶Ⅰ基因的克隆及在酵母中的表达

采用RT—PCR技术,从黑曲霉的菌丝体RNA中扩增出葡萄糖淀粉酶GAI的结构基因,将其连接到pPIC9载体中,转入巴斯德毕赤酵母GSll5中,获得12株Mut＇重组酵母;甲醇诱导目的蛋白分泌表达,葡萄糖淀粉酶酶活最高达180U／ml,占上清液蛋白的38％。通过GAI在巴斯德毕赤酵母中的表达,重点讨论了目的基因拷贝数,基因的偏爱性等对外源基因分泌表达的影响。     

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。     

山梨醇脱氢酶的克隆、表达及活性检测

目的:从氧化葡糖杆菌H24中克隆山梨醇脱氢酶基因进行表达并检测其活性。方法:以氧化葡糖杆菌H24基因组DNA为模板,PCR扩增包括启动子、结构基因及其后的终止序列在内的山梨醇脱氢酶基因;将PCR产物插入pMD18T载体,转化大肠杆菌DH5α;通过活性电泳检测山梨醇脱氢酶在大肠杆菌中的表达及活性。结果:从氧化葡糖杆菌H24中扩增得到山梨醇脱氢酶基因并在大肠杆菌中实现表达,重组菌株经活性电泳检测具有醇糖转化活性。结论:原核表达的山梨醇脱氢酶具有很强的醇糖转化活性。     

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

地衣芽孢杆菌α—淀粉酶基因在枯草芽孢杆菌中的诱导表达

采用PCR技术扩增了sacB基因的启动子-信号序列,并将扩增的序列重组进含地衣芽孢杆菌α-淀粉酶基因的质粒载体上构建了含α-淀粉酶基因的分泌型表达载体pSA60。将pSA60转化枯草芽孢杆菌QB1098后,α-淀粉酶基因在sacB基因启动子-信号序列的调控和蔗糖的诱导下获得表达,表达产物分泌至胞外。     

InFerGene公司集中力量生产食品酶

InFerGene 公司的研究与发展部门将力量集中在重组体 DNA 技术的应用上,以改进用于食品和饮料工业的一些酶的微生物生产。最初,InFerGene 公司主要是生产重组体酵母,而现在主要生产一些如曲霉这种丝状真菌和芽孢杆菌种。最主要的计划是生产葡糖淀粉酶。In-FerGene 公司的一些研究人员已从一种曲霉菌株中分离出这种葡糖淀粉酶基因,他们将这种基因插入一种载体,并用它来转化一种更好的寄主菌株。目的是增加每个发酵罐运转期的葡糖淀粉酶产量,减少发酵罐的运转时间,降低生产成本,以获得一种比较标准和统一的产品。     

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。     

用酵母分泌表达味觉变革蛋白

研究将酸味变成甜味等修饰味觉的蛋白(味觉变革蛋白)的横滨国立大学教育部教授栗原良枝等小组着手研究用基因重组酵母分泌表达味觉变革蛋白奇异果素、葡糖醛酸。奇异果素是与东燃公司,葡糖醛酸是与旭电化工业,东京大学农学部教授荒井综一共同开发的。4月2日在日本农艺化学会上发表了用大肠杆菌表达重组基因的成果,但是用重组大肠杆菌表达的蛋白都没有味觉变革活性。目的是用酵母分泌表达,获得活性型蛋白。为味觉变革蛋白的结构活性的解明和大量生产开辟道     

Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544

We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein. With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library. The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expressed in conidia, while no expression could be detected in mycelia by Northern blot analysis of mycelial RNA. The gene encodes a protein with a calculated molecular mass of 75 kDa that is different from all fructosyltransferases in the databases. The only homology that could be detected was to the invertase of Aspergillus niger (EMBL accession no. L06844). The gene was functionally expressed in Escherichia coli, yeast, and potato plants. With protein extracts from transgenic bacteria and yeast, fructooligosaccharides could be produced in vitro. In transgenic potato plants, inulin molecules of up to 40 hexose units were synthesized in vivo. While in vitro experiments with protein extracts from conidia of Aspergillus sydowi yielded the same pattern of oligosaccharides as extracts from transformed bacteria and yeast, in vivo inulin synthesis with fungal conidia leads to the production of a high-molecular-weight polymer.     

Identification of a nuclear pheromone-sensitive protein kinase not identical to p34CDC28 in Saccharomyces cerevisiae

Abstract A fragment containing the full length cDNA from Aspergillus oryzae α-amylase has been amplified by PCR using specific synthetic oligonucleotides. The amplified cDNA was designed to favour its expression in yeast by modifying its upstream untranslated region. It was subcloned in the expression vector pYEXα1, placed under the control of the yeast CYC1-GAL10 promoter and used to transform Saccharomyces cerevisiae . Cells were then able to express and secrete active α-amylase to the medium in a regulated fashion. The recombinant enzyme had similar electrophoretic mobility and catalytic properties to the original A. oryzae α-amylase.     

Genetic transformation of Trichosporon cutaneum with a plasmid, pAN 7–1, from filamentous fungi

Genetic transformation of a biological oxygen demand (BOD)-sensing yeast, Trichosporon cutaneum IFO 10466 was carried out using a plasmid shuttle vector, pAN 7–1, between Escherichia coli and Aspergillus niger and Aspergillus nidulans. The hygromycin B-resistant transformants were obtained with the plasmid by using the spheroplasts. The transformation frequency was up to 70 colonies per g of plasmid DNA. Southern-blot analysis and transformation test of E. coli using DNA extracts from hygromycin B-resistant transformants of IFO 10466 indicated that pAN 7–1 existed independently in the cells without integration into the chromosome. These results suggest that pAN 7–1 has potential availability for introduction and expression of the external gene such as a bioluminescence gene, lux or luc, to create a luminous yeast for BOD sensing. © Rapid Science Ltd. 1998     

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.     

Expression in a wine yeast strain of the Aspergillus niger abfB gene

Abstract A recombinant wine yeast strain has been constructed expressing the gene coding for a-L-arabinofuranosidase B from Aspergillus niger under the control of the yeast actin gene promoter. The protein is efficiently secreted by the recombinant yeast, allowing its purification and characterisation. The heterologous α-l-arabinofuranosidase B shows similar physico-chemical properties to the native enzyme. The wine produced in microvinification experiments using the recombinant yeast presents the same oenological characteristics as obtained with the untransformed strain. The a-L-arabinofuranosidase B protein is detected throughout the fermentation.     

A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans

An Aspergillus nidulans kinase gene, which encodes a protein kinase with high similarity to mitogen-activated protein kinases involved in cell wall construction and morphogenesis in yeast species, was cloned and sequenced. Targeted deletion of the Aspergillus nidulans kinase gene indicates that this kinase is involved in germination of conidial spores and polarized growth. These defects were largely remedied on complex high osmolarity media, although abnormal swellings of hyphal tips were still observed. Glycerol (1 M) only supported the growth of compact colonies. The Aspergillus nidulans kinase gene is, thus, required for normal polarized growth at several stages of colony formation in the filamentous fungus A. nidulans.     

Recent advances in the molecular genetics of industrial filamentous fungi

Recently, considerable research effort has focused on the molecular genetics of filamentous fungi of industrial importance. Intense research was initiated following reports of transformation systems for the non-commercial filamentous fungi Neurospora crassa and Aspergillus nidulans, and was prompted by two principle considerations: (1) the possibility of exploiting the inherent ability of many filamentous fungi to secrete copious quantities of protein in submerged culture, and (2) the disappointing yields of many heterologous proteins when secreted from prokaryotic and yeast expression systems.