脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF的基因克隆及序列分析

利用氯化苄分别从真菌顶头孢（Cephalosporium acremonium)和产黄头孢（Acremonium chrysogenum)中提取总DNA，通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF，结果只能从黄头孢DNA趸扩增出cefEF基因。测序结果表明，其与已报道的基因序列只有3个碱基的差异，推断的氨基酸序列只有2个氨基酸有差异，并未涉及活性中心。同时表明，国外指所指的与该酶有关的顶头孢（Cephalosporium acremonium或Acremonium chryso-geum)对应的是国内的产黄头孢（Acremonium chrysogenum)。

Cloning, Sequence Analysis of Deacetoxycephalosporin C Synthetase/Hydroxylase Gene cefEF

Chromosomal DNA preparations were made form the fungi C.acremonium and A.chrysogenum respectively by a method using 1\|Chloro methyl benzene solution.Then the cefEF gene of deacetoxycephalosporin C synthetase/hydroxylase was amplified by polymerase chain reaction.The cefEF gene could only be amplified from the chromosomal DNA of A.chrysogenum. This fragment about 1.0 kb was cloned into pGME\|T vector and then sequenced.The sequence indicates that the cloned cefEF gene contains 999 nucleotides encoding for 333 amino acids and there are three bases and two amino acids different from those that have been reported.Both of the two amino acids are not in active site.The result also indecates that the reported C.acremonium or A.chrysogenum generating deacetoxycephalosporin C synthetase/hydroxylase corresponds with A.chrysogenum but not C.acremonium in China.