Barredo J L, Cantoral J M, Alvarez E, Díez B, Martín J F
Area de Microbiología, Facultad de Biología, Universidad de León, Spain.
Mol Gen Genet. 1989 Mar;216(1):91-8. doi: 10.1007/BF00332235.
A gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the amino-terminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the lambda phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position -11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54-1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.
利用与产黄青霉AS-P-78异青霉素N合酶氨基末端对应的探针，将编码该酶的基因（ips）克隆到一个3.9 kb的SalI片段中。SalI片段被剪切成一个1.3 kb的NcoI-BglII片段，该片段包含一个996个核苷酸的开放阅读框，编码一个由331个氨基酸组成、分子量为38012道尔顿的多肽。产黄青霉AS-P-78菌株的ips基因预测的多肽在第195位含有一个酪氨酸，而高产青霉素菌株23X-80-269-37-2的基因在同一位置显示为异亮氨酸。ips基因利用λ噬菌体PL启动子在大肠杆菌小细胞中表达。与顶头孢霉和构巢曲霉ips基因的上游序列相比，在产黄青霉ips基因的上游区域发现了一些相似的序列基序。引物延伸研究表明，mRNA的起始位置与位于保守的富含嘧啶序列中、靠近两个CAAG框的-11位的一个T重合。用ips基因转化的产黄青霉Wis 54-1255克隆显示出比未转化培养物高五倍的异青霉素N合酶活性。
