The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.

The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases. Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines. Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species. The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin-and aclacinomycin-producing Streptomyces strains. The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines. Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway. This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct. The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone.

Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.Most anthracyclines that have been investigated are biosynthesized via aklavinone as a key intermediate. Biosynthesis has been well demonstrated with daunomycins (e.g., see references 8 and 13), which, due to their clinical significance, are the best known anthracyclines. However, nogalamycin (Fig. 1A), produced by Streptomyces nogalater (ATCC 27451), is a distinctive anthracycline. Its aglycone (nogalamycinone) is synthesized via a polyketide biosynthetic pathway from 10 acetates (17). Nogalamycin has two sugar residues: a neutral sugar, nogalose, and a dimethyl amino sugar, nogalamine. The features that make nogalamycin different from most other anthracyclines are the attachment of nogalamine at both C-1 (by a typical glycosidic bond) and at C-2 (by an unusual C-C bond) (16) and the opposite stereochemistry at C-9 (1). In addition to the nogalamycin group, steffimycins are the only anthracyclines known to have the 9S configuration (2).It has not been possible to produce nogalamycinone ( Fig. 1B), either (i) chemically, because nogalamine could not be removed from the aglycone (only the bisanhydro form of the aglycone was obtained by treatment with strong base at elevated temperatures) (16), or (ii) by genetic engineering, due to the lack of the cyclase determining the unique stereochemistry at C-9. In this paper, we report the production of nogalamycinone by genetically engineered Streptomyces lividans TK24, clarifying an important intermediate of nogalamycin biosynthesis.Strains, culture conditions, and DNA manipulation. Streptomyces strains were grown at 30°C in tryptone soya broth containing thiostrepton (50 g/ml) for preparation of plasmid DNA and in E1 medium containing glucose (20 g/liter), soluble starch (20 g/liter), Farmamedia (5 g/liter), yeast extract (2.5 g/liter), CaCO 3 (3 g/liter), NaCl (1 g/liter), MgSO 4 ⅐ 7H 2 O (1 g/liter), and K 2 HPO 4 ⅐ 3H 2 O (1 g/liter) in tap water (pH 7.5) for production of anthracycline metabolites (19). A DNA fragment derived from a nogalamycin biosynthetic cluster containing a cyclase gene was cloned in a pIJ486-based plasmid and introduced into S. lividans TK24 (6). Streptomyces galilaeus mutant H039 producing aklavinone-(rhodinose) 2-3 (19) was used as a host in attempts to produce C-9 stereoisomers of aklavinone. DNA isolation and manipulation were carried out by standard methods (6, 14).Cloning and sequencing of the gene for nogalamycin cyclization. In our attempts to clarify the molecular genetics of nogalamycin biosynthesis, we expanded the previously characterized nogalamycin gene cluster (15,20,21). A fragment from this previously cloned nogalamycin biosynthetic...

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.