The Streptomyces peucetius dpsY and dnrX genes govern early and late steps of daunorubicin and doxorubicin biosynthesis

Abstract

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:73167321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.

FIG. 1

Biosynthesis of DNR and DXR in S. peucetius. Only the key intermediates and genes discussed in the text are shown. Thin and open thick arrows signify single or multiple steps, respectively. For the baumycin-like compounds, the structure of baumycin A1 is illustrated but the C-4 and C-13 positions can be modified in other types of baumycins (39).

FIG. 2

(A) Organization of the DXR biosynthesis and self-resistance genes in the region surrounding dnrX and dpsY. The drrA and drrB resistance genes are described by Guilfoile and Hutchinson (16), and dnmZ is described by Otten et al. (32). B, BamHI; Bg, BglII (B). The DNA and deduced protein sequence of the region from panel A characterized in this paper. Only the restriction stites of interest are shown. Probable translational start and stop codons are doubly and singly underlined, respectively. The fM and asterisk indicate the probable beginning and end of the open reading frame (only the first 10 amino acids of dnmZ are shown).

FIG. 3

Insertional inactivation of the dnrX and dpsY genes. Restriction maps are shown for the BamHI fragment containing the dnrX and dpsY genes (in pWHM279), the disrupted dnrX gene (in pWHM280), and the disrupted dpsY gene (in pWHM282). Arrows indicate the relative directions of transcription of the dnrX, dpsY, and aphII genes. Restriction sites abbreviations: Ba, BamHI; Bg, BglII; Ec, EcoNI; Kp, KpnI; Nc, NcoI; Sp, SphI; Ss, SstI; and Xh, XhoI.