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首页> 外文期刊>Yeast >Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae
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Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

机译：脂单胞菌IGC4052B的第二个α-淀粉酶基因（LKA2）的克隆，鉴定及其在酿酒酵母中的表达

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Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second a-amylase gene (LKA2) from the same strain of L kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55 307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1(p)-LKA2 in S. cerevisiae resulted in the production of biologically active a-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60degreesC. Besides LKA1, LKA2 is only the second L kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities. The nucleotide sequence of the LKA2 gene has been assigned GenBank Accession No. AF443872.
机译：脂单胞菌分泌一系列高效淀粉酶（即α-淀粉酶，葡糖淀粉酶，异淀粉酶和环麦芽糊精葡聚糖转移酶活性），因此被认为是已知的最有效的生淀粉降解酵母之一。以前，我们已经克隆和鉴定了来自L. kononenkoae IGC4052B（CBS5608T）的LKA1α-淀粉酶基因的基因组和cDNA拷贝，并在酿酒酵母和粟酒裂殖酵母中表达了它们。在这里，我们报道了来自克农氏科同一菌株的第二个α-淀粉酶基因（LKA2）的基因组和cDNA副本的克隆和表征。最初将LKA2克隆为1663 bp cDNA，带有1496个核苷酸的开放阅读框（ORF）。 LKA2的序列分析表明，该ORF编码499个氨基酸的蛋白质（Lka2p），预测分子量为55 307 Da。 LKA2编码的α-淀粉酶与几种细菌的环麦芽糊精葡聚糖转移酶以及构巢曲霉，西方脱皮芽孢杆菌，腓肠酵母和Sz的α-淀粉酶显示出显着的同源性。庞贝。当在酿酒酵母中在磷酸甘油酸激酶基因启动子（PGK1（p））的控制下表达LKA2时，发现基因组拷贝包含一个55 bp的内含子，这会削弱异源宿主中生物学活性Lka2p的产生。与基因组拷贝相反，在酿酒酵母中PGK1（p）-LKA2的cDNA构建体的表达导致了生物活性α-淀粉酶的产生。由酿酒酵母产生的LKA2编码的α-淀粉酶对含有α-1，4糖苷键的底物表现出高特异性。发现Lka2p的最适pH为3.5，最适温度为60℃。除LKA1外，LKA2也是酿酒酵母中克隆和表达的第二个L科农科基因。编码这些高效α-淀粉酶的这两个基因的克隆，表征和共表达，是旨在开发酿酒酵母淀粉分解菌株以将淀粉有效地生物转化为商业上重要商品的广泛研究计划的重要组成部分。 LKA2基因的核苷酸序列已被赋予GenBank登录号AF443872。

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