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

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubi cin (DXR) gene cluster that contains dpsH, the dpsG polyketide synthase gen e, the putative dnrU ketoreductase gene, dnrV, and the doxA cytochrome P-45 0 gene. These five genes were characterized by sequence analysis, and the e ffects of replacing dnrU, dnrV, doxA, or dpsH with mutant alleles and of do xA overexpression on the production of the principal anthracycline metaboli tes of S. peucetius were studied. The exact roles of dpsH and dnrV could no t be established, although dnrV is implicated in the enzymatic reactions ca talyzed by DoxA, but dnrU appears to encode a ketoreductase specific for th e C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precurs ors. 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. Colomb o, and C. R. Hutchinson, J. Bacteriol. 180:2379-2386, 1998) double mutant a nd 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 incr ease in DXR production. In contrast, overexpression of dnrV and doxA jointl y in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increa sed the DXR titer 36 to 86%.