RESUMEN
BACKGROUND: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. RESULTS: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. CONCLUSION: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposed.
Asunto(s)
Flavanonas/biosíntesis , Plantas/metabolismo , Streptomyces/metabolismo , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Aciltransferasas/deficiencia , Aciltransferasas/genética , Secuencia de Aminoácidos , Amoníaco-Liasas/química , Amoníaco-Liasas/deficiencia , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Flavanonas/análisis , Flavanonas/química , Genoma Bacteriano , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Fenilalanina/metabolismo , Plantas/química , Alineación de Secuencia , Streptomyces/genética , Tirosina/metabolismoRESUMEN
ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.