头孢菌素C酰基转移酶在顶头孢霉中的表达

以假单胞菌的头孢菌素C(CPC)酰基转移酶为基础,根据顶头孢霉密码子偏爱性及RNA二级结构预测分析,设计并合成了全长2349 bp的CPC酰基转移酶基因ecs,并将其在大肠杆菌中表达.结果证明,表达蛋白能将CPC转化为7-ACA.将ecs表达质粒转化CPC产生菌顶头他霉,通过PCR、Southern blotting以及Western blotting等方法验证,表明ecs基因已成功整合到顶头孢霉染色体上并得到表达,重组菌的发酵也显示部分CPC直接转化成了7-ACA.

Expression of Cephalosporin C Acylase in Acremonium chrysogenum

A 2 349 bp ecs gene was designed based on codon bias of Acremonium chrysogenum and analysis of RNA secondary structure prediction of cephalosporin C(CPC)acylase originated from Pseudomonas N176.The ecs was then synthesized and induced into E.coli BL21(DE3).The results showed that CPC could be converted to 7-ACA by purified recombinant ECS protein.A.chrysogenum expression plasmid containing ecs was transformed into this CPCproducing strain and the integration of heterologous genes was verified by PCR and Southern blotting.The expression of ECS in A.chrysogenum was verified by Western blotting.Fermentation of the transformats showed that a part of CPC had been converted to 7-ACA.