Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris
Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris
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DOI：
10.1002/yea.1243
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发表时间：
2005-07-15
期刊：
影响因子：
2.6
通讯作者：
Torres, FAG
中科院分区：
文献类型：
--
作者：
de Almeida, JRM;de Moraes, LMP;Torres, FAG
本文报道了用聚合酶链式反应的方法从巴斯德毕赤酵母中克隆出3-磷酸甘油酸激酶基因(PGK1)。PGK1基因的编码序列全长1251bp，编码416个氨基酸残基的多肽，与其他酵母的同源蛋白具有很高的同源性。该基因的启动子区域(P-PGK1)含有在其他PGK1基因中发现的调控顺式元件，如TATA盒、富含CT的区块和一个热休克元件。在3‘下游区域，我们发现了一个与转录终止有关的三方元件5’-Tag-TAGT-TTT3‘。与其他酵母一样，巴斯德毕赤酵母的PGK1基因是以单拷贝基因的形式存在的。Northern印迹分析表明，该基因转录为1.5kb的mRNA；当细胞生长在葡萄糖上时，该mRNA的水平比生长在甘油上的细胞高两倍。将枯草芽孢杆菌的α-淀粉酶基因置于P-PGK1的控制下，进一步证实了该基因受碳源的转录调控：与甘油和甲醇相比，葡萄糖对该基因的表达水平更高。与P-PGK1的强度相关的初步结果表明，P-PGK1在巴斯德毕赤酵母中是一种潜在的结构性异源表达的替代物。该基因序列已登录GenBank，登录号为：AY288296。版权所有(C)2005 John Wiley&Sons，Ltd.
We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P-PGK1) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. In the 3' downstream region we identified a tripartite element 5'-TAG-TAGT-TTT3', which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single-copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two-fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the a-amylase gene from Bacillus subtilis was placed under the control of P-PGK1: higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of P-PGK1 show that it represents a potential alternative to constitutive heterologous expression in P. pastoris. The sequence of the gene has been deposited in GenBank under Accession No. AY288296. Copyright (c) 2005 John Wiley & Sons, Ltd.