Gutiérrez S, Velasco J, Fernandez F J, Martín J F
Department of Ecology, Genetics and Microbiology, University of León, Spain.
J Bacteriol. 1992 May;174(9):3056-64. doi: 10.1128/jb.174.9.3056-3064.1992.
The gene (cefG) encoding the acetyl coenzyme A:deacetylcephalosporin C acetyltransferase of Cephalosporium acremonium (synonym Acremonium chrysogenum) C10 has been cloned. It contains two introns and encodes a protein of 444 amino acids with an M(r) of 49,269 that correlates well with the M(r) deduced by gel filtration. The cefG gene is linked to the cefEF gene (encoding the bifunctional deacetoxycephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the cefEF gene. Two transcripts of 1.2 and 1.4 kb were found in C. acremonium that correspond to the cefEF and cefG genes, respectively; the degree of expression of the cefG gene was clearly lower than that of the cefEF gene in 48-h cultures. The cloned cefG complemented the deficiency of deacetylcephalosporin acetyltransferase in the nonproducer mutant C. acremonium ATCC 20371 and restored cephalosporin biosynthesis in this strain. Heterologous expression of the cefG genes took place in Penicillium chrysogenum. The deacetylcephalosporin acetyltransferase showed a much higher degree of homology with the O-acetylhomoserine acetyltransferases of Saccharomyces cerevisiae and Ascobolus immersus than with other O-acetyltransferases. The cefEF-cefG cluster of genes encodes the enzymes that carry out the three late steps of the cephalosporin biosynthetic pathway and is not linked to the pcbAB-pcbC gene cluster that encodes the first two steps of the pathway.
已克隆了顶头孢霉(同义词产黄顶孢霉)C10中编码乙酰辅酶A:去乙酰头孢菌素C乙酰基转移酶的基因(cefG)。它含有两个内含子,编码一个由444个氨基酸组成、相对分子质量为49269的蛋白质,这与凝胶过滤推导的相对分子质量高度相关。cefG基因与cefEF基因(编码双功能去乙酰氧头孢菌素C合酶/羟化酶)相连,但它的表达方向与cefEF基因相反。在顶头孢霉中发现了分别对应于cefEF和cefG基因的1.2 kb和1.4 kb的两种转录本;在48小时培养物中,cefG基因的表达程度明显低于cefEF基因。克隆的cefG弥补了非生产型突变体顶头孢霉ATCC 20371中去乙酰头孢菌素乙酰基转移酶的缺陷,并恢复了该菌株中的头孢菌素生物合成。cefG基因在产黄青霉中进行了异源表达。去乙酰头孢菌素乙酰基转移酶与酿酒酵母和浸没曲霉的O - 乙酰高丝氨酸乙酰基转移酶的同源性比与其他O - 乙酰转移酶的同源性高得多。cefEF - cefG基因簇编码执行头孢菌素生物合成途径最后三个步骤的酶,并且不与编码该途径前两个步骤的pcbAB - pcbC基因簇相连。