title: Construction of recombinant Escherichia coli D11/pMSTO and its use in enzymatic preparation of 7-aminocephalosporanic acid in one pot.
abstract: The main drawback in the industrial production of 7-aminocephalosporanic acid is the accumulation of intermediate (AKA-7-ACA) and destruction of substrate (cephalosporin C) catalyzed by catalase and beta-lactamase. To overcome the adverse effect of these enzymes on the conversion process, Escherichia coli D11 with mutation of katG, katE and ampC genes was constructed by P1 phage transduction, which enabled it not to produce catalase and beta-lactamase, respectively. At the same time, recA mutation in D11 increased the stability of foreign plasmid. With D11 used as host, both d-amino acid oxidase and GL-7-ACA acylase were cloned and expressed by the recombinant plasmids of pMSS or pMSTO, and the production of two enzymes could be increased by addition of 1.0% glucose. Cells of recombinant strain D11/pMSTO could directly convert cephalosporin C into 7-aminocephalosporanic acid at 25 degrees C, with the yield of more than 74%. The data suggested that the constructed D11/pMSTO could be an alternative catalyst for production of 7-aminocephalosporanic acid in one pot.
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310029, China.
authors.forename: Huabao
authors.initials: H
authors.lastname: Zheng
authors.forename: Tongbo
authors.initials: T
authors.lastname: Zhu
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Zhao
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Yang
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D002511:Cephalosporins / Q000096:biosynthesis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014161:Transduction, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; C081063:glutaryl-7-aminocephalosporanic acid; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; C102664:hydroperoxidase II; D002374:Catalase; C051253:katG protein, E coli; D001618:beta-Lactamases