title: Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster.
abstract: The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.
authors: Microbiology Section, Department of Molecular Biology, Faculty of Biology and Environmental Sciences, Vegazana Campus, University of León, León 24071, Spain.
authors: Y
authors: Y
authors: Martínez-Burgo
authors: Microbiology Section, Department of Molecular Biology, Faculty of Biology and Environmental Sciences, Vegazana Campus, University of León, León 24071, Spain.
authors: R
authors: R
authors: Álvarez-Álvarez
authors: Institute of Biotechnology INBIOTEC, Av. Real 1, León 24006, Spain.
authors: R
authors: R
authors: Pérez-Redondo
authors: Microbiology Section, Department of Molecular Biology, Faculty of Biology and Environmental Sciences, Vegazana Campus, University of León, León 24071, Spain. Electronic address: paloma.liras@unileon.es.
authors: P
authors: P
authors: Liras
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D002513:Cephamycins / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D018014:Gene Transfer Techniques; D008826:Microbial Sensitivity Tests; D005810:Multigene Family / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002513:Cephamycins; D004274:DNA, Recombinant; C024925:cephamycin C
keywords: CcaR regulatory protein; Cephamycin C; Heterologous expression; Streptomyces clavuligerus; Streptomyces flavogriseus