RESUMEN
The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for their biological activity, being responsible for specific contacts to molecular targets and significantly affecting their pharmacokinetic properties. In order to provide tools for the targeted alteration of natural product glycosylation patterns, significant strides have been made to understand the biosynthesis of activated deoxysugars and their transfer. We report here efforts towards the production of plasmid-borne biosynthetic gene cassettes capable of producing TDP-activated forms of D-mycaminose, D-angolosamine and D-desosamine. We additionally describe the transfer of these deoxysugars to macrolide aglycones using the glycosyl transferases EryCIII, TylMII and AngMII, which display usefully broad substrate tolerance.
Asunto(s)
Glucosamina/análogos & derivados , Macrólidos/química , Macrólidos/metabolismo , Clonación Molecular , Ingeniería Genética , Glucosamina/química , Glucosamina/metabolismo , Estructura Molecular , Familia de Multigenes/genética , Análisis de Secuencia , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
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 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 spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.
Asunto(s)
ADN/química , Insecticidas/química , Macrólidos/química , Sintasas Poliquetidas/química , Tetranychidae/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Eritromicina/química , Escherichia coli/metabolismo , Ivermectina/análogos & derivados , Ivermectina/química , Modelos Moleculares , Ingeniería de Proteínas , Saccharopolyspora/enzimología , Saccharopolyspora/metabolismo , Streptomyces/enzimología , Streptomyces/metabolismoRESUMEN
The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.