顶头孢霉乙酰转移酶基因的克隆、表达和活性研究

Cloning expression and activity analysis of DAC-acetyltransferanse gene from Acremonium chrysogenum

头孢菌素C是工业上制备半合成β-内酰胺类抗生素的重要原料。头孢菌素C生物合成途径中的最后一步是将脱乙酰头孢菌素C在乙酰转移酶的作用下形成头孢菌素C,编码催化这一步骤的乙酰转移酶的基因是cefG。由于cefG在染色体上具有内含子,通过RT-PCR的方法从头孢菌素产生菌顶头孢霉总RNA中获得了约1.1kb的cefG基因,将cefG基因克隆到大肠埃希菌质粒pET28a进行表达,SDS-PAGE电泳显示有明显的条带,体外活性测试表明表达产物能将脱乙酰头孢菌素C转化成头孢菌素C。

Cephalosporin C （CPC） is a clinically important raw material for β-lactam semi-synthesis. The final step, deacetylcephalosporin C （DAC） transformed to CPC, in the biosynthesis pathway of CPC is catalyzed by acetyhransferase encoded by cefG. Since cefG is a gene with two introns in Acremonium chrysogenum genome, RT-PCR was performed to obtain - 1.1kb cefG from the total RNA of CPC producing strain. The resulting product was cloned into Escherchia coli expressing vector pET28a for acetyltransferase expression. After IPTG induction, an apparent polyketide band was observed on the SDS-PAGE electrophoresis. The bioactivity of the recombinant protein was tested in vitro, and the result showed that DAC was partly transformed into CPC.