大肠杆菌头孢菌素C乙酰酯酶的敲除对头孢菌素C酰化酶应用的影响

The Effect of Cephalosporin C Acetyl Esterase Knockout in Escherichia coli on the Application of Cephalosporin C Acylase

7-氨基头孢烷酸(7-ACA)是合成头孢菌素类抗生素的重要中间体,工业上通常采用头孢菌素C酰化酶一步水解头孢菌素C制备,但在该反应产物中存在一个主要杂质3-去乙酰基-7-氨基头孢烷酸(D-7-ACA),该杂质的产生是由大肠杆菌中内源基因aes编码的头孢菌素C乙酰酯酶水解头孢菌素C或7-ACA引起的。为了防止D-7-ACA的形成,获得高品质7-ACA,减少下游精制成本,采用大肠杆菌双质粒pTargetF/pCas敲除系统,设计相应的gRNA以及同源修复供体DNA,对大肠杆菌BL21(DE3)中的aes进行敲除,从而获得了Aes缺陷型菌株BL21(DE3)△aes。将携带头孢菌素C酰化酶(CPCacy)表达质粒pET30-CPCacy的重组工程菌BL21(DE3)/pET30-CPCacy和BL21(DE3)△aes/pET30-CPCacy所诱导表达的孢菌素C酰化酶细胞破碎上清液进行7-ACA的生产对比试验,结果表明,敲除菌BL21(DE3)△aes/pET30-CPCacy对头孢菌素C的转化率为98.8%而原始菌为98.5%,同时7-ACA的产率为80.7%而原始菌株为80.2%,杂质D-7-ACA的产率仅为0.1%,为原始菌的25%,这些工作为进一步生产高品质的7-ACA奠定了基础。

7-aminocephalosporanic acid(7-ACA)is an important intermediate for synthesis of cephalosporin antibiotics,which is produced by enzymatic conversion of cephalosporin C using cephalosporin C acylase in industry.However,during the reaction process,there is a major impurity 3-deacetyl-7-aminocephalosporanic acid(D-7-ACA)generated from the degradation of cephalosporin C or 7-ACA by cephalosporin C acetyl esterase encoded by the aes gene of Escherichia coli.In order to obtain high-quality 7-ACA and reduce downstream refining costs,it is necessary to prevent the formation of D-7-ACA.Therefore,the corresponding gRNA and donor DNA fragments were designed and the gene aes was knocked out from the chromosome of E.coli BL21(DE3)to generate the engineer E.coli BL21(DE3)△aes using the pTargetF/pCas knockout system.Then,the plasmid of pET30-CPCacy was constructed by inserting the gene CPCacy encoding cephalosporin C acylase into the backbone of pET30(a).The cell lysis supernatants of recombinant strains expressing the cephalosporin C acylase plasmids,including E.coli BL21(DE3)/pET30-CPCacy and E.coli BL21(DE3)△aes/pET30-CPCacy,were applied to the production of the 7-ACA.During the process of cephalosporin C bioconversion,the cephalosporin C utilization efficiency,the yield of 7-ACA and impurity D-7-ACA by each engineered strain were compared.The cephalosporin C conversion rate was 98.8%in E.coli BL21(DE3)△aes/pET30-CPCacy and 98.5%in the original strain,respectively.At the same time,the yield of 7-ACA was 80.7%while that of the original strain was 80.2%,and the yield of impurity D-7-ACA was only 0.1%which was a quarter of the original strain.This work would lay a foundation for the further production of high-quality 7-ACA.