中国生物工程杂志

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.