title: Double knockout of β-lactamase and cephalosporin acetyl esterase genes from Escherichia coli reduces cephalosporin C decomposition.
abstract: 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, β-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(ΔampC), E. coli JM105(Δaes) and E. coli JM105(ΔampC, Δaes). The CPC decomposition was reduced to 12.2% in E. coli JM105(Δaes), 1.3% in E. coli JM105(ΔampC), and even undetectable in ampC-aes double knockout cells of E. coli JM105(ΔampC, Δaes). When catalyzed by crude MBP-sCPCAcy isolated from E. coli JM105(ΔampC, Δaes)/pMKC-sCPCacy (3377U·l(-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, ΔampC, Δaes)/pET28-sCPCacy.
authors.affiliation: Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, P R China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Luo
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D058619:Maltose-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D002265:Carboxylic Ester Hydrolases; C019861:cephalosporin-C deacetylase; D001618:beta-Lactamases