Gutiérrez S, Díez B, Montenegro E, Martín J F
Department of Ecology, Genetics and Microbiology, University of León, Spain.
J Bacteriol. 1991 Apr;173(7):2354-65. doi: 10.1128/jb.173.7.2354-2365.1991.
A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha-aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI-BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin-biosynthetic genes.
通过与产黄青霉的pcbAB和pcbC基因杂交，克隆了顶头孢霉C10 DNA的一个24 kb区域。该区域的一个3.2 kb BamHI片段互补了顶头孢霉N2突变体结构pcbC基因中的突变，从而产生头孢菌素。一个功能性的α-氨基己二酰-半胱氨酰-缬氨酸（ACV）合成酶由一个15.6 kb的EcoRI - BamHI DNA片段编码，这通过产黄青霉ACV合成酶缺陷型突变体的互补得以证明。通过分别用pcbC和pcbAB基因内部的探针进行Northern（RNA印迹）杂交，发现了1.15 kb和11.4 kb的两种转录本。在pcbC基因上游定位了一个11136 bp的开放阅读框，它与11.4 kb转录本的起始和终止区域匹配。它编码一个3712个氨基酸的蛋白质，推导的Mr为414791。该基因的核苷酸序列与编码产黄青霉ACV合成酶的pcbAB基因显示出62.9%的相似性；两种ACV合成酶中54.9%的氨基酸是相同的。顶头孢霉ACV合成酶的推导氨基酸序列中出现了三个高度重复区域。每个区域都类似于产黄青霉ACV合成酶推导序列中的三个重复结构域，也类似于短短芽孢杆菌的短杆菌肽合成酶I和杀念菌素合成酶I的氨基酸序列。这些区域可能对应于ACV合成酶蛋白中的氨基酸活化结构域。此外，两种真菌的ACV合成酶中都存在一个硫酯酶结构域。在多酶非核糖体肽合成酶以及聚酮和脂肪酸合成酶中存在的结构域之间发现了相似性。pcbAB基因与pcbC基因相连，形成了一个早期头孢菌素生物合成基因簇。
