RESUMO
Expression of the aranciamycin biosynthetic gene cluster in Streptomyces diastatochromogenes Tü6028 resulted in production of four novel compounds, aranciamycins E, F, G, and H with different decorations in the tetracyclic backbone. Two derivatives contain a D-amicetose moiety at C7 (aranciamycins F and G), two are hydroxylated at position C1 (aranciamycins E and G), and one is hydroxylated at C13 (aranciamycin F). Analysis of the biological activities of the aranciamycins against two human tumor cell lines--MCF-7 and MATU--shows surprising impact of the hydroxyl group at position C1 on activity. As aranciamycins E and G were the most active derivatives, hydroxylation of the C1 appears to coincide with increased antitumor activity of aranciamycins.
Assuntos
Antraciclinas/química , Antraciclinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Engenharia Genética , Streptomyces/metabolismo , Sequência de Aminoácidos , Antraciclinas/metabolismo , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Streptomyces/química , Streptomyces/genética , Relação Estrutura-AtividadeRESUMO
The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.
Assuntos
Produtos Biológicos , Genômica , Preparações Farmacêuticas , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , Espectroscopia de Ressonância Magnética , Programas de Rastreamento , Dados de Sequência Molecular , Estrutura Molecular , Homologia de Sequência de AminoácidosAssuntos
Antraciclinas/metabolismo , Glicosiltransferases/biossíntese , Glicosiltransferases/genética , Inibidores de Proteases , Antraciclinas/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , DNA Bacteriano/genética , Dados de Sequência Molecular , Família Multigênica , Plasmídeos/genética , Inibidores de Proteases/farmacologia , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
We have cloned the gene that encodes a novel glucosyl transferase (AraGT) involved in rhamnosylation of the polyketide antibiotic Aranciamycin in Streptomyces echinatus. AraGT comprises two domains characteristic of bacterial glycosyltranferases. AraGT was synthesized in E. coli as a decahistidinyl-tagged polypeptide. Purified AraGT is dimeric, displays a T(mapp) of 30 degrees C and can glycosylate the aglycone of an Aranciamycin derivative as shown by liquid chromatography and mass spectrometry. The availability of functional AraGT will allow the generation Aranciamycin-based combinatorial libraries.
Assuntos
Antraciclinas/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/isolamento & purificação , Sequência de Aminoácidos , Cromatografia Líquida , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
The glycosyltransferase LanGT2 is involved in the biosynthesis of the hexasaccharide side chain of the angucyclic antibiotic landomycin A. Its function was elucidated by targeted gene inactivation of lanGT2. The main metabolite of the obtained mutant was identified as tetrangulol (4), the progenitor of the landomycin aglycon (7). The lack of the sugar side chain indicates that LanGT2 catalyzes the priming glycosyl transfer in the hexasaccharide biosynthesis: the attachment of a D-olivose to O-8 of the polyketide backbone. Heterologous expression of urdGT2 from S. fradiae Tü2717 in this mutant resulted in the production of a novel C-glycosylated angucycline (6).