RESUMO
Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6'-deamino-6'-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.
Assuntos
Expressão Gênica , Técnicas Genéticas , Ribostamicina/análogos & derivados , Ribostamicina/biossíntese , Vias Biossintéticas/genética , Genes Bacterianos/genética , Família Multigênica/genética , Ribostamicina/química , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/genéticaRESUMO
Aminoglycosides are a class of important antibiotic compounds used for various therapeutic indications. In recent times, their efficacy has been curtailed due to the rapid development of bacterial resistance. There is a need to develop novel derivatives with an improved spectrum of activity and higher sensitivity against pathogenic bacteria. Although efforts have been focused on the development of newer therapeutic agents by chemical synthesis, to our knowledge, there has been no attempt to harness the potential of microorganisms for this purpose. Escherichia coli affords a widely studied cellular system that could be utilized not only for understanding but also for attempting to engineer the biosynthetic pathway of secondary metabolites. The primary metabolic pathway of E. coli can be engineered to divert the precursor pool required for the biosynthesis of secondary metabolites. Utilizing this approach previously, we engineered E. coli host and generated E. coli M1. Here, we produced a ribostamycin derivative in the engineered host by heterologous expression of the recombinants constructed from the genes encoding the biosynthetic pathway in aminoglycoside-producing strains. The products obtained from the transformants were isolated, analyzed and verified to be ribostamycin derivatives. The study further demonstrated the importance of E. coli as surrogate antibiotic producer and also offered future possibility for the production of other aminoglycoside derivatives through genetic engineering and expression in a heterologous background.
Assuntos
Antibacterianos/biossíntese , Escherichia coli/genética , Ribostamicina/análogos & derivados , Sequência de Aminoácidos , Aminoglicosídeos/biossíntese , Antibacterianos/metabolismo , Vias Biossintéticas , Clonagem Molecular , Descoberta de Drogas , Farmacorresistência Bacteriana , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Bacterianos , Engenharia Genética , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/metabolismo , Ribostamicina/biossíntese , Ribostamicina/metabolismo , Transformação BacterianaRESUMO
A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.
Assuntos
Sulfato de Butirosina/biossíntese , Família Multigênica , Ribostamicina/biossíntese , Streptomyces/genética , Acetilação , Aminoglicosídeos/metabolismo , Sulfato de Butirosina/metabolismo , Cromatografia Líquida de Alta Pressão , Farmacorresistência Bacteriana/genética , Modelos Biológicos , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
By the use of our improved colony selection technique, xylostasin and ribostamycin producing mutants were isolated from nitrosoguanidine treated Bacillus circulans B15M, a producer of butirosins A and B. Among these structurally related aminoglycosides, ribostamycin is the well-known product of a Steptomyces and has not been isolated as a bacterial metabolite. A selected mutant of strain 306, which produces xylostasin and ribostamycin, was futher mutagenized in expectation of getting an improved strain having the ability to accumulate a large amount of ribostamycin in the culture broth. One mutant, strain 451, derived from strain 306, produced ribostamycin free of xylostasin.
Assuntos
Antibacterianos/biossíntese , Bacillus/metabolismo , Sulfato de Butirosina/biossíntese , Ribostamicina/biossíntese , Bactérias/efeitos dos fármacos , Cromatografia em Camada Fina , Fermentação , Testes de Sensibilidade Microbiana , Mutação , Ribostamicina/farmacologiaRESUMO
A mutant of a neomycin-producting Streptomyces fradiae was found which synthesizes ribostamycin instead of neomycin. After a reverse mutation new colonies were obtained producting neomycin again. Ribostamycin might thus be considered as an intermediate in the biosynthesis of neomycin.