Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine

Abstract

Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis.Daunorubicin (DNR) and its C-14-hydroxylated derivative doxorubicin (DXR) (Fig. 1) are clinically important antitumor agents, and like many microbial secondary metabolites, they require a deoxyhexose component for their biological activity (7). These deoxyhexose constituents are commonly 6-deoxyhexoses including 2,6-and 4,6-dideoxy or trideoxy amino hexoses, as is the case for DNR and DXR, which contain the 2,3,6-trideoxy-3-aminohexose daunosamine. The biosynthesis of these biologically important deoxy sugars is not well understood, but progress is being made, fueled in part by the availability of DNA sequence data. These data facilitate the construction of mutant strains that are disrupted in potential sugar genes and permit the assignment of reasonable functions to deduced gene products by sequence comparisons to proteins of known function, such as those for the biosynthesis of rhamnose (16) and the 3,6-dideoxyhexoses (14) that are found in the lipopolysaccharides of gram-negative bacteria. Thus a putative glucose-1-phosphate thymidylyltransferase gene, dnmL (formerly dnrL) (8), which presumably governs the first step of daunosamine biosynthesis (Fig. 1), has been identified in the DNR gene cluster of the wild-type Streptomyces peucetius ATCC 29050 strain, and a thymidine-diphospho (TDP)-glucose-4,6-dehydratase for the formation of the 4-keto-6-deoxyhexulose nucleotide that is a key precursor in deoxyhexose biosynthesis has been purified (28). The gene encoding the putative TDP-glucose-4,6-dehydratase has been localized outside of the DNR gene cluster (8), while a TDP-glucose-4,6-dehydratase homolog, dnmM (formerly dnrM) (8), found adjacent to dnmL, is apparently nonfunctional due to a frameshift mutation. Inactivation of the dnmJ (formerly dnrJ) gene (17) indicates that it is required for daunosamine biosynthesis, and the similarity between DnmJ and AscC (29) suggests that DnmJ is likely to be involved in the addition of the C-3 amino group to a daunosamine precursor (30). Furthermore, disruptions of the dnmT (24) and dnmQ (formerly dnrQ) (21) genes indicate that they too are required for daunosamine biosynthesis although their enzymatic functions have not been established. In this report we extend our investigation of TDP-daunosamine biosynthesis through DNA sequencing and characterization of the dnmZ, dnmU, and dnmV genes that encode a protein of unknown function plus the putative TDP-4-keto-6-deoxyglucose-3(5)-epimerase and TDP-4-ketodeoxyhexulose reductase, respectively, of the daunosamine biosynthetic pathw...

Identification and Expression of Genes Involved in Biosynthesis of l -Oleandrose and Its Intermediate l -Olivose in the Oleandomycin Producer Streptomyces antibioticus

“…Comparison of the deduced product of this gene with proteins in databases showed clear similarities with various dTDP-4-keto-6-deoxyglucose 3,5-epimerases involved in 6-deoxysugar biosynthesis in the streptomycin pathway by Streptomyces glaucescens (50% identity; PIR accession no. S44236), the StrM epimerase from S. griseus, which is also involved in streptomycin biosynthesis (50% identity) (40), the EryBVII protein from the erythromycin pathway from S. erythraea (42.4% identity) (16,53), and the DnmU protein from the daunorubicin pathway in S. peucetius (42.8% identity) (37). OleL also resembles different RfbC proteins involved in lipopolysaccharide biosynthesis in Mycobacterium tuberculosis (7) and Shigella flexneri (30).…”

“…However, the biochemical pathways involved in 6-deoxyhexose biosynthesis have not been characterized in most cases. Several authors have reported the cloning of different genes involved in the biosynthesis of 6-deoxyhexoses in antibiotic and antitumor pathways: streptomycin (40), erythromycin (10,16,46,53), tylosin (15,19,31), daunosamine (36,37), lincomycin (38), mithramycin (28), methymycin-pikromycin (62), and granaticin (4,22). Macrolide antibiotics contain mono-or disaccharides attached to the macrolactone rings which are important for activity.…”

“…The oleU gene would code for a polypeptide of 295 amino acids and an estimated M r of 32,098. The OleU deduced product displays similarity to a large family of sugar reductases involved in antibiotic biosynthesis: StrL from the streptomycin pathway in S. griseus (57.4% identity) (40), DnmV from the daunorubicin pathway in S. peucetius (27.8% identity) (37), and EryBIV from the erythromycin pathway in S. erythraea (24.8% identity) (16,53). It also resembles the well-characterized UDP-galactose 4-epimerase encoded by the galE gene of E. coli (3).…”

Biosynthesis of Polyketides in Heterologous Hosts

“…Sometimes, however, cosubstrate availability can be an issue. For example, glycosyltransferases associated with polyketide pathways often utilize specialized TDP-deoxysugars, themselves the products of multistep biosynthetic pathways (26,66,80). As with PKS genes, the clustering of the genes responsible for TDP-sugar biosynthesis facilitates their expression in heterologous hosts (64).…”

The dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene

“…The high-copy-number Streptomyces shuttle vector pWHM3 was from Vara et al (1989), and the low-copynumber shuttle vector pWHM601 was from Guilfoile & Hutchinson (1991). pWHM219 was from Otten et al (1997), pWHM533 was from Otten et al (1995), pWHM1104 was from Tang et al (1996), and pWHM289 and pWHM298 were from Lomovskaya et al (1999). pMT3000 and the promoter probe vector pMT3003.1 (Paget et al, 1994) were provided by C. P. Smith (University of Manchester, UK).…”