Molecular cloning and expression of the phenoxazinone synthase gene from Streptomyces antibioticus.
DNA fragments from the Streptomyces antibioticus genome have been cloned in Streptomyces lividans using the SphI site of the vector, pIJ702. Using a sib selection procedure, transformants were screened for their ability to produce levels of the enzyme, phenoxazinone synthase, which were detectable by enzyme assay. Three clones were obtained by this procedure. The recombinant plasmids from these clones contained inserts of 1.77, 2.45, and 4.28 kilobases in size. The clones and the corresponding recombinant plasmids were, therefore, referred to as PHS 1.8, PHS 2.4 and PHS 4.3. PHS 2.4 produced transformants with significantly higher phenoxazinone synthase specific activities than those observed in comparably grown S. antibioticus. Southern blotting showed that the cloned 2.45-kilobase insert hybridized to a fragment of exactly the same size in an SphI digest of S. antibioticus total DNA. Using a streptomycete-coupled transcription-translation system, PHS 2.4 was shown to code for the synthesis of a protein with the electrophoretic and immunological properties of the phenoxazinone synthase subunit. Thus, the 2.45-kilobase insert represents the structural gene for that subunit. The protein produced by S. lividans transformed with PHS 2.4 was shown to be virtually identical to the S. antibioticus subunit in terms of its electrophoretic mobility, peptide profile, and amino acid composition. It was further observed that cloning of the 2.45-kilobase insert in the orientation opposite to that obtained originally still resulted in high levels of phenoxazinone synthase expression in transformed S. lividans. However, the repression of phenoxazinone synthase synthesis by glucose which has been observed in S. antibioticus was not found when PHS 2.4 was used to transform S. lividans.
D000596:Amino Acids / Q000032:analysis; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D013302:Streptomyces / Q000201:enzymology*; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D000596:Amino Acids; D010088:Oxidoreductases; C023944:2-aminophenol oxidase; D004262:DNA Restriction Enzymes