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PMID: 9864344 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Journal of bacteriology ·Vol. 181 ·No. 1 ·1999-01-00 ·Pages 305-18

Lomovskaya N, Otten SL, Doi-Katayama Y, Fonstein L, Liu XC, Takatsu T, Inventi-Solari A, Filippini S, Torti F, Colombo AL, Hutchinson CR

Abstract

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%.

MeSH Terms
Acyl Carrier Protein/genetics,metabolism Alcohol Oxidoreductases/genetics,metabolism Amino Acid Sequence Anthracyclines/metabolism Bacterial Proteins/genetics,metabolism Base Sequence Chromosome Mapping Cloning, Molecular Cytochrome P-450 Enzyme System/genetics,metabolism DNA Primers/genetics DNA, Bacterial/genetics Doxorubicin/biosynthesis Gene Expression Genes, Bacterial Genetic Complementation Test Mixed Function Oxygenases/genetics,metabolism Molecular Sequence Data Multigene Family Mutagenesis, Insertional Mutation Streptomyces/enzymology,genetics,metabolism Substrate Specificity
Chemicals
Acyl Carrier Protein Anthracyclines Bacterial Proteins DNA Primers DNA, Bacterial DnrV protein, Streptomyces peucetius Doxorubicin Cytochrome P-450 Enzyme System Mixed Function Oxygenases Alcohol Oxidoreductases daunorubicin ketoreductase doxA protein, Streptomyces peucetius
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Lomovskaya N
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706, USA.
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
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.181.1.305-318.1999
Published
1999-01-00
Pages
305-18
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC103563
Subset
IM
Grants
NCI NIH HHS · CA 64161 · United States
Databases
GENBANK
U77891
External Links
PubMed source DOI: 10.1128/JB.181.1.305-318.1999 PMC Full Text View journal details
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