Double knockout of 尾-lactamase and cephalosporin acetyl esterase genes from Escherichia coli reduces cephalosporin C decomposition

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• 作者：Ying Wang¹ ; Huimin Yu¹ ; ^{yuhm@tsinghua.edu.cn} ; Jing Zhang¹ ; Hui Luo² ; Zhongyao Shen¹
• 关键词：CPC acylase ; CPC decomposition ; 尾-Lactamase ; Cephalosporin acetyl esterase ; Genes double knockout ; CPC utilization efficiency
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2012
• 期刊代码：pc9_13891723
• 类别：ce
• 出版时间：June, 2012
• 卷：113
• 期：6
• 页码：737-741
• 文件大小：484 K

摘要

The phenomenon of CPC decomposition occurs in Escherichia coli JM105/pMKC-sCPCacy during the one-step enzymatic conversion of cephalosporin C (CPC) into 7-aminocephalosporanic acid (7-ACA) by CPC acylase (sCPCAcy) for synthesis of cephalosporin antibiotics. E. coli JM105/pMKC-sCPCacy can constitutively produce sCPCacy as a fusion protein with maltose binding protein (MBP). Control experiments verified that the cell lysis solution from the host E. coli JM105 resulted in CPC decomposition by approximately 15%. Two miscellaneous enzymes, 尾-lactamase (AmpC) and cephalosporin acetyl esterase (Aes), are believed to play a major role in the degradation of CPC. Using the Red recombination system, the genes ampC, aes or both ampC and aes were knocked out from the chromosome of E. coli JM105 to generate the engineers: E. coli JM105(螖ampC), E. coli JM105(螖aes) and E. coli JM105(螖ampC, 螖aes). The CPC decomposition was reduced to 12.2%in E. coli JM105(螖aes), 1.3%in E. coli JM105(螖ampC), and even undetectable in ampC-aes double knockout cells of E. coli JM105(螖ampC, 螖aes). When catalyzed by crude MBP-sCPCAcy isolated from E. coli JM105(螖ampC, 螖aes)/pMKC-sCPCacy (3377 U路l^鈭?#xA0;1), the CPC utilization efficiency increased to 98.4%from the original 88.7%. Similar results were obtained for the ampC-aes double knockout host derived from E. coli JM109(DE3) and the CPC utilization efficiency enhanced to 99.3%in the catalysis of crude sCPCAcy harvested from E. coli JM109(DE3, 螖ampC, 螖aes)/pET28-sCPCacy.