RESUMEN
Novamethymycin (9), a novel macrolide antibiotic, was isolated from Streptomyces venezuelae, the producer of methymycin (4) and neomethymycin (5). Spectroscopic analysis of 9 indicated that it is highly related to 4 and 5 but contains hydroxy groups at both C-10 and C-12 on the macrolactone ring. Bioconversion studies using the PikC cytochrome P450 hydroxylase demonstrated that 4 is converted to 9, further broadening the remarkable substrate flexibility of this enzyme.
Asunto(s)
Antibacterianos/aislamiento & purificación , Macrólidos , Streptomyces/química , Antibacterianos/química , Antibacterianos/farmacología , Bacillus subtilis , Catálisis , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de Fourier , Streptomyces/metabolismo , Relación Estructura-ActividadRESUMEN
The Streptomyces venezuelae pikD gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. venezuelae, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying pikD restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. venezuelae wild-type and pikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, and desI but not those controlling pikRI or pikC.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Genes Reguladores , Macrólidos , Streptomyces/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Amino Azúcares , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
In Streptomyces venezuelae, four polyketide synthase (PKS) polypeptides encoded by pikAI-pikAIV are used to generate 10 and 12-membered macrocyclic structures, narbonolide and 10-deoxymethynolide. Sequence analysis suggests these genes are translationally coupled with downstream genes, pikAV (encoding a type II thioesterase), desVIII-desVI (encoding enzymes responsible for production of the final glycosylated products pikromycin, narbomycin, methymycin and neomethymycin) and desR (a resistance gene). Type II thioesterases have been suggested to have an editing function in polyketide biosynthesis and deletion of the corresponding genes often leads to decreased levels of polyketide production. Surprisingly an in-frame deletion of 687 bp of the 843 bp pikAV ORF led to a strain SC1022 that produced normal yields of polyketide products, but only in the aglycone form. Plasmid-based expression of the desVIII-VI and desR in the SC1022 strain completely restored production of glycosylated products, despite the absence of a functional pikAV gene product. Under these conditions the PikAV TEII therefore does not play an important role in polyketide biosynthesis, and its function remains an enigma. These observations also demonstrate that the region of pikAV DNA deleted in strain SC1022 contains a transcription unit essential for expression of the des genes. A sequence alignment of PikAV with members of the highly conserved type II thioesterases revealed a short divergent region at the carboxy terminus, suggesting a region of pikAV that might contain such a transcription unit. DNA containing this region of pikAV was shown to be able to increase plasmid-based expression of both crotonyl CoA reductase gene (ccr) and the erythromycin resistance gene (ermE) in S. venezuelae.
Asunto(s)
Ácido Graso Sintasas/genética , Streptomyces/genética , Tioléster Hidrolasas/genética , Acil-CoA Deshidrogenasas , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Cósmidos/genética , ADN Recombinante , Ácido Graso Sintasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Glicosilación , Lactonas/química , Lactonas/metabolismo , Macrólidos/metabolismo , Datos de Secuencia Molecular , Mutación , Operón , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Streptomyces/enzimología , Streptomyces/metabolismo , Tioléster Hidrolasas/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Pikromycin-related macrolides have recently attracted significant research interest because they are structurally related to the semisynthetic ketolide antibiotics that have demonstrated promising potential in combating multi-drug-resistant respiratory pathogens. Cloning and in-depth studies of the pikromycin biosynthetic gene cluster from Streptomyces venezuelae have led to new avenues in modular polyketide synthases, deoxysugar biosynthesis, cytochrome P450 hydroxylase, secondary metabolite gene regulation, and antibiotic resistance. Moreover, the knowledge and tools used for these studies are proving to be valuable in the development of advanced technologies for combinatorial biosynthesis of new macrolide antibiotics. This review summarizes these new developments and introduces S. venezuelae as a powerful new system for secondary metabolite pathway engineering from bench-top genetic manipulation to product fermentation.
Asunto(s)
Antibacterianos/biosíntesis , Macrólidos , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Carbohidratos , Técnicas Químicas Combinatorias , Ingeniería Genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Streptomyces/enzimologíaRESUMEN
BACKGROUND: . Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain. RESULTS: . Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro. CONCLUSIONS: . These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Genes Bacterianos/genética , Macrólidos/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Medios de Cultivo , Cinética , Datos de Secuencia Molecular , Plásmidos/genéticaRESUMEN
In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.