https://hdl.handle.net/10259/4862 2026-10-12T10:38:19Z https://hdl.handle.net/10259/12152 The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis Mendes, Marta V.; Tunca, Sedef; Antón Fidalgo, Nuria; Recio, Eliseo; Sola-Landa, Alberto; Aparicio, Jesús F.; Martín, Juan F. The biosynthesis of the antifungal pimaricin in Streptomyces natalensis is very sensitive to phosphate regulation. Concentrations of inorganic phosphate above 1 mM drastically reduced pimaricin production. At 10 mM phosphate, expression of all the pimaricin biosynthesis (pim) genes including the pathway-specific positive regulator pimR is fully repressed. The phoU-phoR-phoP cluster of S. natalensis encoding two-component Pho system was cloned and sequenced. Binding of the response regulator PhoP to the consensus PHO boxes in the phoU-phoRP intergenic promoter region was observed. A phoP-disrupted mutant and a phoR-phoP deletion mutant were obtained. Production of pimaricin in these two mutants increased up to 80% in complex yeast extract–malt extract (YEME) or NBG media and showed reduced sensitivity to phosphate control. Four of the pim genes, pimS1, pimS4, pimC and pimG showed increased expression in the phoP-disrupted mutant. However, no consensus PHO boxes were found in the promoter regions of any of the pim genes, suggesting that phosphate control of these genes is mediated indirectly by PhoR-PhoP involving modification of pathway-specific regulators. 2007-01-01T00:00:00Z https://hdl.handle.net/10259/12150 Cholesterol Oxidases Act as Signaling Proteins for the Biosynthesis of the Polyene Macrolide Pimaricin Mendes, Marta V.; Recio, Eliseo; Antón Fidalgo, Nuria; Guerra, Susana M.; Santos-Aberturas, Javier; Martín, Juan F.; Aparicio, Jesús F. The gene cluster responsible for pimaricin biosynthesis in Streptomyces natalensis contains a cholesterol oxidase-encoding gene (pimE) surrounded by genes involved in pimaricin production. Gene-inactivation and -complementation experiments revealed that pimE encodes a functional cholesterol oxidase and, surprisingly, that it is also involved in pimaricin biosynthesis. This extracellular enzyme was purified from S. natalensis culture broths to homogeneity, and it was shown to restore pimaricin production when added to the mutant culture broths. Other cholesterol oxidases also triggered pimaricin production, suggesting that these enzymes could act as signaling proteins for polyene biosynthesis. This finding constitutes the description of a cholesterol oxidase gene with an involvement in antibiotic biosynthesis, and it broadens the scope of the biological functions for this type of oxidase. 2007-03-01T00:00:00Z https://hdl.handle.net/10259/12148 Characterization of the polyene macrolide P450 epoxidase from Streptomyces natalensis that converts de-epoxypimaricin into pimaricin Mendes, Marta V.; Antón Fidalgo, Nuria; Martín, Juan F.; Aparicio, Jesús F. The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD. Pimaricin is derived from its immediate precursor de-epoxypimaricin by epoxidation of the C-4–C-5 double bond on the macrolactone ring. We have overproduced PimD with a N-terminal His6 affinity tag in Escherichia coli and purified the enzyme for kinetic analysis. The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 nm, indicating that it is a cytochrome P450. Purified PimD was shown to catalyse the in vitro C-4–C-5 epoxidation of 4,5-de-epoxypimaricin to pimaricin. The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 °C. The kcat value for the epoxidation of de-epoxypimaricin was similar to the values reported for other macrolide oxidases. Enzyme activity was inhibited at high substrate concentration. This is the first time that a polyene macrolide P450 mono-oxygenase has been expressed heterologously and studied. The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics. 2005-01-01T00:00:00Z https://hdl.handle.net/10259/12107 PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis Antón Fidalgo, Nuria; Santos Aberturas, Javier; Mendes, Marta V.; Guerra, Susana M.; Martín, Juan F.; Aparicio, Jesús F. Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix–turn–helix (HTH) motif of the LuxR type. Gene replacement of pimM from the Streptomyces natalensis chromosome with a mutant version lacking the HTH DNA-binding domain resulted in complete loss of pimaricin production, suggesting that PimM is a positive regulator of pimaricin biosynthesis. Complementation of the ΔpimM mutant with a single copy of pimM integrated into the chromosome restored pimaricin production. The insertion of a single copy of pimM, with its own promoter, into the S. natalensis wild-type strain boosted pimaricin production. Gene expression analyses in S. natalensis wild-type and ΔpimM by reverse transcriptase PCR (RT-PCR) of the pimaricin gene cluster revealed the targets for the PimM regulatory protein. According to these analyses, the genes responsible for initiation and first elongation cycles of polyketide chain extension are among the major targets for regulation. Other pim genes are differentially affected. Interestingly, our results indicate that PimM plays its regulatory role independently of PimR, the first pathway-specific regulator of pimaricin biosynthesis. 2007-09-01T00:00:00Z