Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene.

Lomovskaya, N; Otten, S L; Doi-Katayama, Y; Fonstein, L; Liu, X C; Takatsu, T; Inventi-Solari, A; Filippini, S; Torti, F; Colombo, A L; Hutchinson, C R

Lomovskaya, N; Otten, S L; Doi-Katayama, Y; Fonstein, L; Liu, X C; Takatsu, T; Inventi-Solari, A; Filippini, S; Torti, F; Colombo, A L; Hutchinson, C R.

Afiliación

Lomovskaya N; School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706, USA.

J Bacteriol ; 181(1): 305-18, 1999 Jan.

Article en En | MEDLINE | ID: mdl-9864344

RESUMEN

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that contains dpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and the doxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacing dnrU, dnrV, doxA, or dpsH with mutant alleles and of doxA overexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379-2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316-7321, 1996) triple mutant. Overexpression of doxA in a doxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxA jointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.

Asunto(s)

Oxidorreductasas de Alcohol/genética; Oxidorreductasas de Alcohol/metabolismo; Proteínas Bacterianas/genética; Proteínas Bacterianas/metabolismo; Sistema Enzimático del Citocromo P-450/genética; Sistema Enzimático del Citocromo P-450/metabolismo; Doxorrubicina/biosíntesis; Genes Bacterianos; Oxigenasas de Función Mixta/genética; Oxigenasas de Función Mixta/metabolismo; Streptomyces/genética; Streptomyces/metabolismo; Proteína Transportadora de Acilo/genética; Proteína Transportadora de Acilo/metabolismo; Secuencia de Aminoácidos; Antraciclinas/metabolismo; Secuencia de Bases; Mapeo Cromosómico; Clonación Molecular; Cartilla de ADN/genética; ADN Bacteriano/genética; Expresión Génica; Prueba de Complementación Genética; Datos de Secuencia Molecular; Familia de Multigenes; Mutagénesis Insercional; Mutación; Streptomyces/enzimología; Especificidad por Sustrato

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Streptomyces / Proteínas Bacterianas / Doxorrubicina / Sistema Enzimático del Citocromo P-450 / Oxidorreductasas de Alcohol / Genes Bacterianos / Oxigenasas de Función Mixta Idioma: En Revista: J Bacteriol Año: 1999 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

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