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 designated as pCSE10000.
Cephalexin 반합성 효소의 유전자를 박테리아에서 E. coli HB101에 pBR322를 사용해 옮겼다. 여기서 획득한 유전자는 다음 다섯 가지 박테리아 중의 한가지에서 유래하였다. 그 다섯가지 박테리아는 Acetobacter turbidans ATCC 9325, Bacillus megaterium ATCC 14945, Kluyvera citrophila KY 7844, Pseudomonas melanogenum IFO 12020, and Xanthomonas citri IFO 3835이다.
염색체 DNA를 이들 박테리아에서 얻어서 Pst I 제한효소로 부분적으로 처리했다. pBR322 DNA도 Pst I 제한효소로 처리하고 calf intestinal alkaline phosphatase로 탈인산화하였다. Pst I 제한효소로 처리된 염색체 DNA를 pBR322에 연결시켜 E. coli HB101에 형질 전환시켰다. Cephalexin 반합성 효소의 유전자를 가진 크론을 분석 하기위해 Staphylococcus aureus ATCC 6538P를 사용해 생물학적 분석방법을 확립했다.
12,000여개의 형질전환균을 시험한 결과 2개의 형질전환균에 cephalexin 반합성 효 소의 유전자를 갖고 있다고 나타났다. 이들 두개의 형질전환균에서 플라스미드를 얻어 제한효소로 처리한 결과 똑같은 플라스미드로 밝혀졌다.
이 cephalexin 반합성 효소를 가진 새로운 재조합 플라스미드를 pCSE22000 으로 표시했다. pCSE22000 플라스미드의 크기가 22Kb인데 이것의 크기를 줄여 10Kb인 pCSE10000을 얻어냈다.
