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 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%.
Lomovskaya
N
N
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706, USA.
Otten
S L
SL
Doi-Katayama
Y
Y
Fonstein
L
L
Liu
X C
XC
Takatsu
T
T
Inventi-Solari
A
A
Filippini
S
S
Torti
F
F
Colombo
A L
AL
Hutchinson
C R
CR
D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D018943:Anthracyclines / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004317:Doxorubicin / Q000096:biosynthesis*; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D009154:Mutation; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D018943:Anthracyclines; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C116904:DnrV protein, Streptomyces peucetius; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; C116921:daunorubicin ketoreductase; C116920:doxA protein, Streptomyces peucetius