RESUMO
Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Quinonas/química , Quinonas/metabolismo , Streptomyces/metabolismo , Aminobenzoatos/metabolismo , Antibióticos Antineoplásicos/química , Proteínas de Bactérias/metabolismo , Benzoquinonas , Clonagem Molecular , Hidroxibenzoatos , Lactamas Macrocíclicas , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Família Multigênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Rifabutina/análogos & derivados , Análise de Sequência de DNA , Streptomyces/genéticaRESUMO
Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.
Assuntos
Proteínas de Bactérias/genética , Teste de Complementação Genética , Engenharia Genética/métodos , Quinonas/metabolismo , Recombinação Genética , Streptomyces/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/enzimologia , Bacteriófago lambda/genética , Benzoquinonas , Conjugação Genética , DNA Bacteriano/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Deleção de Genes , Lactamas Macrocíclicas , Plasmídeos , Quinonas/química , Streptomyces/metabolismo , Fatores de TempoRESUMO
Two new geldanamycin derivatives produced by genetic engineering of Streptomyces hygroscopicus strain K309-27-1 were isolated and characterized. Removal of the 8-methyl group of geldanamycin was achieved by replacing the AT4 domain of the polyketide synthase with a malonyl AT domain. The resulting strain produced 8-demethyl geldanamycin (2) and 4,5-epoxy-8-demethylgeldanamycin (3). The structures of both molecules were elucidated through interpretation of 1D and 2D NMR data as well as comparison with authentic geldanamycin derivatives. Compounds 2 and 3 displayed moderate cytotoxicity against the human breast cancer cell line SK-BR-3.
Assuntos
Antibióticos Antineoplásicos/isolamento & purificação , Engenharia Genética , Quinonas/síntese química , Quinonas/isolamento & purificação , Streptomyces , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Benzoquinonas , Linhagem Celular Tumoral , Humanos , Lactamas Macrocíclicas , Estrutura Molecular , Quinonas/química , Quinonas/farmacologia , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.
Assuntos
Antineoplásicos , Engenharia Genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Policetídeo Sintases/genética , Quinonas , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sequência de Bases , Benzoquinonas , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas , Conformação Molecular , Dados de Sequência Molecular , Policetídeo Sintases/metabolismo , Quinonas/isolamento & purificação , Quinonas/metabolismo , Quinonas/farmacologia , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602.