Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.
The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.
Binnie
C
C
International Process Development Group, Pfizer Limited, Sandwich, Kent, United Kingdom.
Warren
M
M
Butler
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics*; D010118:Oxytetracycline / Q000096:biosynthesis*; D010957:Plasmids; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D010105:Oxygenases; C034689:anhydrotetracycline oxygenase; D010118:Oxytetracycline