The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures.

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.

A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin.

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.

Hybrid anthracyclines from a genetically engineered Streptomyces galilaeus mutant.

The genetic engineering of antibiotic-producing Streptomyces strains is an approach that is emerging and ready to become established as a successful methodology in developing analogues of the original, pharmaceutically important, natural products obtained from the organisms. The current report highlights this succes by demonstrating the high-level production of novel anthracyclines. The biosynthetic pathways of the nogalamycin-producing Streptomyces nogalater and the aclacinomycin-producing S. galilaeus were combined by transferring the genes of S. nogalater polyketide synthetase into a nonproducing S. galilaeus mutant. The resulting anthracycline antibiotics that were produced possessed structural features characteristic of compounds from both of the undoctored Streptomycesstrains.

Identification of a cyclase gene dictating the C-9 stereochemistry of anthracyclines from Streptomyces nogalater.

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.

An efficient approach for screening minimal PKS genes from Streptomyces.

Degenerated oligonucleotide primers were designed to amplify fragments of ketosynthase genes from polyketide antibiotics producing Streptomyces spp. and bacterial strains enriched from soil samples. Cell lysates were used as templates in amplification, so time-consuming DNA purification was avoided. A phylogenetic tree constructed from the amino acid sequences of the amplified fragments shows a distribution of spore pigments and antibiotics in separate classes. In addition, several different subgroups form within the antibiotics group. Anthracyclines were divided into separate branches according to the starter unit used in biosynthesis.

Folding of the polyketide chain is not dictated by minimal polyketide synthase in the biosynthesis of mithramycin and anthracycline.

BACKGROUND: Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. RESULTS: In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. CONCLUSIONS: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

Isolation and characterization of 8-demethoxy steffimycins and generation of 2,8-demethoxy steffimycins in Streptomyces steffisburgensis by the nogalamycin biosynthesis genes.

Streptomyces steffisburgensis (NRRL 3193, ATCC 27466) is described as a steffimycin producer. Steffimycin belongs to the anthracycline group of aromatic polyketide antibiotics. The structural analysis of the products accumulated by the wild type ATCC 27466 strain revealed three different forms of 8-demethoxy steffimycin suggesting the loss of C-8 hydroxylation/methylation activity. In our approach to generate new anthracycline molecules, we used this strain as a host in gene cloning. The genes encoding the polyketide ketoreductase and aromatase enzymes of nogalamycin biosynthesis caused the production of 2-demethoxy steffimycins in S. steffisburgensis.

Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis.

The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster.

Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes.

A cluster of anthracycline biosynthetic genes isolated from Streptomyces nogalater was expressed in Streptomyces lividans and in Streptomyces galilaeus. A 12 kb DNA fragment cloned from this cluster in pIJ486 caused the production of a novel compound when introduced into S. lividans. The compound is derived from nogalonic acid methyl ester, an early intermediate in nogalamycin biosynthesis. Complementation with the cloned 12 kb fragment of S. galilaeus mutants blocked in aclacinomycin biosynthesis caused the production of hybrid anthracyclines. Cloning of the nogalamycin gene cluster should make possible a detailed study of the biosynthesis of this interesting antibiotic, as well as the production of novel anthracyclines of potential value as cytostatic drugs.

A gene cluster involved in nogalamycin biosynthesis from Streptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway.

We have analyzed an anthracycline biosynthesis gene cluster from Streptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred to Streptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant of S. nogalater. The S. galilaeus mutants carrying the S. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product of snoA seems to be needed for expression of at least the genes for the minimal PKS.

Hybrid anthracycline antibiotics: production of new anthracyclines by cloned genes from Streptomyces purpurascens in Streptomyces galilaeus.

A DNA segment cloned from Streptomyces purpurascens ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase caused S. galilaeus ATCC 31615 to produce new anthracyclines. When transformed with certain sub-clones of this segment, the host produced glycosides of epsilon-rhodomycinone, beta-rhodomycinone, 10-demethoxycarbonylaklavinone and 11-deoxy-beta-rhodomycinone in addition to those of aklavinone, the natural anthracyclines of S. galilaeus. The first two compounds are S. purpurascens products and the other two are novel compounds that conceptually are structural hybrids between S. galilaeus and S. purpurascens products. Three glycosides of one of the novel aglycones, 11-deoxy-beta-rhodomycinone, were purified and found to possess cytotoxic activity against L1210 mouse leukaemia cells. Separate regions of the cloned S. purpurascens DNA are responsible for modification of the S. galilaeus host product at the 10- and 11-positions.

Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants.

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.