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

Abstract

~~~~~ ~~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).

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

“…Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46). It is postulated that ε-rhodomycinone is glycosylated by condensation with TDP-daunosamine (3,8,23,38,42,44) to form the first glycoside intermediate, here named rhodomycin D, which is then converted by a series of reactions to daunorubicin and doxorubicin. Previously, however, the order of the reactions and the enzymes catalyzing some of the steps in the conversion of rhodomycin D to doxorubicin were not known.…”

Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

“…strain C5 fermentations, very little daunomycin is actually produced ; E-rhodomycinone, a known intermediate, is the major metabolite accumulated (McGuire et al, 1980;Bartel et al, 1990). The fact that CMT activity in crude cell extracts is below the limits of detection correlates with the low daunomycin titres obtained.…”

“…The KA values for carminomycin and 13-dihydrocarminomycin seem to reflect accurately the low concentrations of daunomycin produced by Streptomyces sp. strain C5 (McGuire et al, 1980;Bartel et al, 1990).…”

“…The first half of the pathway, i.e. the formation of 8-rhodomycinone from the theoretical polyketide precursor, has now been characterized in relative detail (Eckardt & Wagner, 1988;Bartel et al, 1990;Connors et al, 1990a;Strohl & Connors, 1992). On the other hand, except for our characterization of TDP-D-glucose 4,6-dehydratase, the first enzyme in TDP-daunosamine biosynthesis (Thompson et al, 1992), and our preliminary description of carminomycin methyltransferase (CMT) activity (Connors et al, 1990b), the enzymic reactions involved in glycosylation and the conversion of 8-rhodomycinone to daunomycin remain largely uncharacterized.…”

“…strain C5 based on results obtained with blocked mutants (Bartel et al, 1990), in vitro bioconversion reactions (Connors et al, 1990a), and the discovery of an enzyme capable of methylating carminomycin (Connors et al, 1990b). The first half of the pathway, i.e.…”