Biosynthesis of Daunorubicin Glycosides: Analysis With Blocked Mutants

1996

J Bacteriol

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located between the daunomycin polyketide biosynthesis gene cluster and a dnrI (transcriptional activator) homolog, revealed the presence of a gene encoding a P-450-like enzyme with a deduced M r of 46,096. Expression of this gene, named herein doxA, in Streptomyces lividans TK24 resulted in in vivo bioconversion of daunomycin to doxorubicin. DoxA showed specificity for only daunomycin and 13-dihydrodaunomycin, both of which were converted to doxorubicin. Daunomycinone (daunomycin aglycone), carminomycin, 13-dihydrocarminomycin, idarubicin, and aklavin were not apparent substrates for DoxA. In vector controls or in vectors in which doxA was poorly expressed, S. lividans catalyzed the reduction of daunomycin and other 13-oxo-anthracyclines and -anthracyclinones to their 13-dihydro homologs.

mentioning

confidence: 87%

Isolation and characterization of a gene from Streptomyces sp. strain C5 that confers the ability to convert daunomycin to doxorubicin on Streptomyces lividans TK24

Dickens

1996

J Bacteriol

“…Colonies of SC5-24 are blue due to production of maggiemycin (McGuire et al, 1980b), a putative pathway shunt product (Eckardt & Wagner, 1988). Mutants which produced yellow or red pigments were chosen from survivors grown on R2YE or NDM agar medium.…”

Section: Mutagenesismentioning

confidence: 99%

Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin biosynthesis

Batel

Connors

Journal of General Microbiology

1990

~~~~~ ~~Streptomyces sp. strain C5, an organism that normally produces baumycins, daunomycin and E-rhodomycinone, was treated with N-methyl-N'-nitro-N-nitrosoganidine (NTG). Mutants blocked at various points in daunomycin and baumycin production were isolated by screening for altered pigmentation and absence of bioactivity against Staphyfococcus aureus. Examination of the mutants by thin-layer chromatography of their accumulated anthracycline metabolites, by cosynthesis assays, and by extract feeding experiments allowed a classification into six groups. These were: dauA, strains that accumulated no anthracyclines but with other blocked mutants cosynthesized anthracyclines (polyketide-synthase-minus mutants); dauG, regulatory mutants that, either alone or mixed with other blocked mutants, accumulated no anthracyclines; d a d , mutants that accumulated aklanonic acid; dauE, mutants that accumulated maggiemycin; dauF, mutants that accumulated aklavinone; and dauH, mutants that accumulated only E-rhodomycinone. Mutant SC5-24 (dauE), which accumulated the shunt product maggiemycin, was re-mutagenized with NTG to obtain blocked mutants in preceding biosynthetic steps; the three groups of double mutants obtained accumulated aklanonic acid (dauC,E), aklanonic acid methyl ester (dauD,E) and aklaviketone (dauE,F).

“…The function of DauH in daunomycin biosynthesis in Streptomyces sp. strain C5 is not known, although it could theoretically catalyze either the condensation of ε-rhodomycinone and TDP-daunosamine to form the hypothesized intermediate rhodomycin (27) or, alternatively, it might encode the enzyme that converts daunomycin to baumycin A1/A2 (23). Streptomyces peucetius DnrS was proposed to encode TDP-daunosamine:ε-rhodomycinone glycosyltransferase, although unequivocal evidence for its role is still lacking (24).…”

mentioning

confidence: 99%

Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5

Dickens

1996

J Bacteriol

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, -rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity.