Molecular cloning of a gene coding for cephalexin semisynthesizing enzyme

Abstract

A gene encoding cephalexin semisynthesizing enzyme was transferred from a bacterial strain to Escherichia coli HB101 by molecular cloning using pBR322 as a vector. It is obvious that the cloned gene is originated from one of the following five strains, e.g. Acetobacter turbidans ATCC 9325, Bacillus megaterium ATCC 14945, Kluyvera citrophila KY 7844, Pseudomonas melanogenum IFO 12020, and Xanthomonas citri IFO 3835. The chromosomal DNA was isolated from these strains and partially digested with Pst I. The pBR322 vector was digested with Pst I and dephosphorylated by calf intestinal alkaline phosphatase. The restriction fragments of chromosomal DNA were joined into the linearized pBR322 DNA and then transformed into E. coli HB101 cells using chloride method. To screen the positive clones containing the DNA fragments coding for cephalexin semisynthesizing enzyme, a bioassay using Staphylococcus aureus ATCC 6538P as a indicating strain was selected. Among the 12,000 colonies tested, two colonies showed clear haloes indicating that these clones are producing cephalexin. The plasmids from two clones were identical as determined by agarose gel electrophoresis after the treatment with restriction endonuclease. The new hybrid plasmid containing cephalexin semisynthesizing enzyme was designated as pCSE2200. To reduce the size of the inserted DNA, pCSE22000 was subjected so subclone. The resulting plasmid was disignated as pCSE10000.