RESUMO
Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.
Assuntos
Família Multigênica/genética , Nogalamicina/biossíntese , Streptomyces/genética , Antibióticos Antineoplásicos/biossíntese , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Glicosilação , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Plasmídeos/genética , Análise de Sequência de DNA , Especificidade da Espécie , Streptomyces/metabolismoRESUMO
Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.
Assuntos
Antibióticos Antineoplásicos/biossíntese , Isomerases/genética , Nogalamicina/biossíntese , Streptomyces/genética , Sequência de Aminoácidos , Antibióticos Antineoplásicos/química , Proteínas de Bactérias , Clonagem Molecular , Meios de Cultura , DNA Fúngico/análise , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Isomerases/biossíntese , Isomerases/metabolismo , Dados de Sequência Molecular , Nogalamicina/química , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster.