Díez B, Mellado E, Rodríguez M, Bernasconi E, Barredo J L
Laboratorio de Ingeniería Genética, Antibióticos S.A., León, Spain.
Appl Microbiol Biotechnol. 1999 Aug;52(2):196-207. doi: 10.1007/s002530051509.
The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (bleR) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pchC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum.
已从产黄青霉中分离出编码依赖NADP的谷氨酸脱氢酶活性的gdhA基因，并对其在基因表达中的应用进行了表征。测定了一个2816 bp基因组片段的核苷酸序列，显示一个1600 bp的开放阅读框被两个分别为160 bp和57 bp的内含子打断，具有真菌共有剪接位点连接。预测的氨基酸序列显示与谷氨酸脱氢酶具有高度同一性，尤其是与构巢曲霉（82%）和粗糙脉孢菌（78%）的谷氨酸脱氢酶。发现gdhA基因在几种产青霉素能力不同的产黄青霉菌株的基因组中以单拷贝存在。分析了gdhA启动子在真菌和大肠杆菌中用于同源和异源基因表达的情况。通过构建与大肠杆菌lacZ基因和印度斯坦链霉菌博来霉素抗性决定簇（bleR）的基因融合体来确定异源基因表达。通过使用产黄青霉的青霉素生物合成基因pchC和penDE以及产黄顶头孢霉的头孢菌素生物合成基因cefEF和cefG来显示同源基因表达。
