Cloning of the enomycin structural gene from Streptomyces mauvecolor and production of recombinant enomycin in Escherichia coli.
A genomic DNA library from the enomycin (ENM) producer, Streptomyces mauvecolor, was screened for the ENM structural gene (enm) by use of a segment of the phenomycin gene (phm) as the probe, and a plasmid, pENI, was constructed. By primer-walking along the insert, a 573 bp DNA sequence that contain an ORF corresponding to preENM was determined. The deduced amino acid composition of ENM was close to that previously reported (MIZUNO, S.; K. NITTA & H. UMEZAWA: Mode of action of enomycin, an antitumor antibiotic of high molecular weight. I. Inhibition of protein synthesis. J. Biochem. 61: 373 approximately 381, 1967). The producer cells expressed enm during an ENM-productive fermentation. An enm-expression plasmid, pENE 1, was constructed, with which E. coli AD202 was transformed. The transformant produced a fusion protein consisting of glutathione-S-transferase (GST) and ENM. The genetically engineered ENM (rENM) inhibited the growth of Hela cells in vitro. Comparison of the base sequence spanning enm with that spanning phm showed that the structural genes were conserved more extensively than were the flanking regions, though the genes were unlikely to be essential to the lives of the producers.
Takeuchi
S
S
Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Oka
T
T
Sakata
N
N
S-Tuchiya
K
K
Hayashi
H
H
Hori
M
M
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010455:Peptides; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013302:Streptomyces / Q000235:genetics*
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D010455:Peptides; D011994:Recombinant Proteins; C012305:enomycin