Cloning and characterization of the isopenicillin N synthase gene of Streptomyces griseus NRRL 3851 and studies of expression and complementation of the cephamycin pathway in Streptomyces clavuligerus.
A gene, pcbC, encoding the isopenicillin N synthase of Streptomyces griseus NRRL 3851, has been cloned in a 6.4-kb Bg/II DNA fragment and located in an internal 1.55-kb PvuII segment by hybridization with the Penicillium chrysogenum pcbC gene. Hybridization studies revealed the presence of homologous sequences in the DNAs of several Streptomyces strains and Nocardia lactamdurans. The S. griseus pcbC gene was not expressed in Streptomyces lividans but was expressed in Streptomyces clavuligerus and complemented a mutation, nce2, that impaired isopenicillin N synthase and cephamycin biosynthesis. The pcbC gene contained an open reading frame of 990 nucleotides that encodes a protein of 329 amino acids with a deduced Mr of 37,371. The isopenicillin N synthase formed after expression of the pcbC gene in the S. clavuligerus nce2 mutant strain was found to have an Mr of 38,000 by gel filtration. A protein of about 38 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of extracts of a transformant of the nce2 mutant strain; this protein was absent from the untransformed mutant strain. The G+C content of the pcbC gene was 63.6%, and the strongly biased codon usage was typical of that of Streptomyces strains. A transcription initiation site was found 44 nucleotides upstream of the ATG translation initiation triplet. A transcript of 1.1 kb was observed in the donor S. griseus strain and also in the S. clavuligerus nce2 mutant strain transformed with the pcbC gene, suggesting that it is transcribed as a monocistronic mRNA.
García-Domínguez
M
M
Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, Spain.
Liras
P
P
Martín
J F
JF
D000192:Actinomycetales / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002461:Cell Line, Transformed; D002513:Cephamycins / Q000096:biosynthesis / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D015698:Genomic Library; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000235:genetics*; D010408:Penicillium chrysogenum / Q000235:genetics; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D002513:Cephamycins; D004247:DNA; D010088:Oxidoreductases; C038370:isopenicillin N synthetase