RESUMEN
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
Asunto(s)
Vías Biosintéticas/genética , Evolución Molecular , Variación Genética , Familia de Multigenes , Bioingeniería , Sintasas Poliquetidas/genética , Sirolimus/química , Sirolimus/metabolismoRESUMEN
A new octaketide named fogacin (1) was isolated from Streptomyces sp. (strain Tü 6319). Furthermore two shunt metabolites, SEK4b (2) and anhydroSEK4b (3), were detected and identified as non-enzymatically cyclized products of polyketide intermediates built during the biosynthesis of actinorhodin. SEK4b (2) as well as anhydroSEK4b (3) were previously described as metabolites of genetically engineered strains.
Asunto(s)
Antibacterianos/química , Macrólidos/química , Streptomyces/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Dicroismo Circular , Macrólidos/aislamiento & purificación , Macrólidos/farmacología , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Rotación Óptica , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría UltravioletaRESUMEN
Hormaomycin produced by Streptomyces griseoflavus is a structurally highly modified depsipeptide that contains several unique building blocks with cyclopropyl, nitro, and chlorine moieties. Within the genus Streptomyces, it acts as a bacterial hormone that induces morphological differentiation and the production of bioactive secondary metabolites. In addition, hormaomycin is an extremely potent narrow-spectrum antibiotic. In this study, we shed light on hormaomycin biosynthesis by a combination of feeding studies, isolation of the biosynthetic nonribosomal peptide synthetase (NRPS) gene cluster, and in vivo and in vitro functional analysis of enzymes. In addition, several nonnatural hormaomycin congeners were generated by feeding-induced metabolic rerouting. The NRPS contains numerous highly repetitive regions that suggest an evolutionary scenario for this unusual bacterial hormone, providing new opportunities for evolution-inspired metabolic engineering of novel nonribosomal peptides.
Asunto(s)
Proteínas Bacterianas/genética , Depsipéptidos/biosíntesis , Péptido Sintasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Depsipéptidos/química , Familia de Multigenes , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Transducción de Señal , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.
Asunto(s)
Benzoquinonas/farmacología , Productos Biológicos/farmacología , Ingeniería Genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Benzoquinonas/química , Benzoquinonas/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
The macrocyclic compound hormaomycin has been investigated by NMR spectroscopy and by restrained molecular-dynamics simulations. Measurement of residual dipolar couplings induced by dissolving the depsipeptide in a polyacrylamide gel compatible with DMSO and their incorporation into the structure calculation of the title compound improved the precision of the family of structures. In DMSO the macrocyclic ring shows two beta-turns, whose positions in the sequence differ from those found in the CDCl3 solution structure and in the crystal structure obtained from hexylene glycol/H2O (50:50). The bulky side chain consisting of a 3-(2-nitrocyclopropyl)alanine and a chlorinated N-hydroxypyrrole moiety is flexible in DMSO.