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

Abstract

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.

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

“…After incubation of the cultures at 30ЊC in a rotary shaker (250 rpm) for 48 h, 100 g of filter-sterilized anthracycline in distilled water were added to the cultures. The cultures containing substrate were then incubated further as above for 24 to 72 h, depending on the experiment, after which the whole culture broth was brought to a pH of 8.5 with 5 N NaOH and extracted with chloroform-methanol (9:1) as previously described (14). For isolation of anthracyclines containing free carboxyl groups, the broth was brought to a pH of 2.5 with 6 N HCl and extracted with n-butanol (21).…”

“…The dauP (15), dauK (15), and doxA (14) genes from the Streptomyces sp. strain C5 anthracycline biosynthesis gene cluster ( Fig.…”

“…For isolation of anthracyclines containing free carboxyl groups, the broth was brought to a pH of 2.5 with 6 N HCl and extracted with n-butanol (21). The bioconversion products were analyzed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and MS as described recently (14). Bioconversion products were compared to authentic standards cochromatographed or run in parallel.…”

“…For MS analysis, 500 g of anthracycline substrate was incubated for 48 h with 48-h-old cultures of recombinant S. lividans grown in YEME medium. The bioconversion products were extracted as described previously, with the exception that the compounds were back-extracted with double-distilled water (14). Dried samples were resuspended in 500 l of methanol, from which 50 l was removed for HPLC and TLC analysis and the remainder was redried for MS analysis.…”

“…We recently showed that the doxA gene of Streptomyces sp. strain C5 encoded a cytochrome P-450 enzyme that was capable of conferring on Streptomyces lividans TK24 the ability to convert daunorubicin to doxorubicin (14). In the present work, we describe the enzymatic activities from Streptomyces sp.…”