Members of the aureolic acid family are tricyclic polyketides with antitumor activity which are produced by different streptomycete species. These members are glycosylated compounds with two oligosaccharide chains of variable sugar length. They interact with the DNA minor groove in high-GC-content regions in a nonintercalative way and with a requirement for magnesium ions. Mithramycin and chromomycins are the most representative members of the family, mithramycin being used as a chemotherapeutic agent for the treatment of several cancer diseases. For chromomycin and durhamycin A, antiviral activity has also been reported. The biosynthesis gene clusters for mithramycin and chromomycin A(3) have been studied in detail by gene sequencing, insertional inactivation, and gene expression. Most of the biosynthetic intermediates in these pathways have been isolated and characterized. Some of these compounds showed an increase in antitumor activity in comparison with the parent compounds. A common step in the biosynthesis of all members of the family is the formation of the tetracyclic intermediate premithramycinone. Further biosynthetic steps (glycosylation, methylations, acylations) proceed through tetracyclic intermediates which are finally converted into tricyclic compounds by the action of a monooxygenase, a key event for the biological activity. Heterologous expression of biosynthetic genes from other aromatic polyketide pathways in the mithramycin producer (or some mutants) led to the isolation of novel hybrid compounds.

Mithramycin is an antitumor compound produced by Streptomyces argillaceus that has been used for the treatment of several types of tumors and hypercalcaemia processes. However, its use in humans has been limited because its side effects. Using combinatorial biosynthesis approaches, we have generated seven new mithramycin derivatives, which either differ from the parental compound in the sugar profile or in both the sugar profile and the 3-side chain. From these studies three novel derivatives were identified, demycarosyl-3D-β-d-digitoxosyl-mithramycin SK, demycarosyl-mithramycin SDK and demycarosyl-3D-β-d-digitoxosyl-mithramycin SDK, which show high antitumor activity. The first one, which combines two structural features previously found to improve pharmacological behavior, was generated following two different strategies, and it showed less toxicity than mithramycin. Preliminary in vivo evaluation of its antitumor activity through hollow fiber assays, and in subcutaneous colon and melanoma cancers xenografts models, suggests that demycarosyl-3D-β-d-digitoxosyl-mithramycin SK could be a promising antitumor agent, worthy of further investigation.

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.

Chromomycin A3 is a member of the aureolic acid group family of antitumour drugs. Three tailoring modification steps occur during its biosynthesis affecting the sugar moieties: two O-acetylations and one O-methylation. The 4-O-methylation in the 4-O-methyl-D-oliose moiety of the disaccharide chain is catalysed by the cmmMIII gene product. Inactivation of this gene generated a chromomycin-non-producing mutant that accumulated three unmethylated derivatives containing all sugars but differing in the acylation pattern. Two of these compounds were shown to be substrates of the methyltransferase as determined by their bioconversion into chromomycin A2 and A3 after feeding these compounds to a Streptomyces albus strain expressing the cmmMIII gene. The same single membrane-bound enzyme, encoded by the cmmA gene, is responsible for both acetyl transfer reactions, which convert a relatively inactive compound into the bioactive chromomycin A3. Insertional inactivation of this gene resulted in a mutant accumulating a dideacetylated chromomycin A3 derivative. This compound, lacking both acetyl groups, was converted in a two-step reaction via the 4E-monoacetylated intermediate into chromomycin A3 when fed to cultures of S. albus expressing the cmmA gene. This acetylation step would occur as the last step in chromomycin biosynthesis, being a very important event for self-protection of the producing organism. It would convert a molecule with low biological activity into an active one, in a reaction catalysed by an enzyme that is predicted to be located in the cell membrane.

Chromomycin A 3 (Fig. 1A) is an antitumor drug produced by Streptomyces griseus and other streptomycete species. It belongs to the class of antitumor compounds of the aureolic acid family (22), which inhibit growth and multiplication of several tumor cell lines. The antitumor properties are ascribed to their inhibitory effects on replication and transcription processes during macromolecular biosynthesis by interacting, in the presence of Mg 2ϩ , with GC-rich nucleotide sequences located in the minor groove of DNA. In this respect, they have been shown to prevent resistance to other antitumor agents by a number of mechanisms, including downregulation of proteins, such as MDR1 (16, 28). Chromomycin and the closely related compound mithramycin were also found to stimulate K562 cell erythroid differentiation (3), to be potent inhibitors of neuronal apoptosis (7), and to have antiviral activity against human immunodeficiency virus type 1 (2).Structurally, chromomycin A 3 is related to mithramycin.Both consist of a tricyclic chromophore (aglycone) with two aliphatic side chains attached at C-3 and C-7. However, they differ in glycosylation pattern. In chromomycin, the carbohydrate moieties and the acetyl groups which decorate these sugars are major structural contributors to the biological activity. Thus, it has been shown that the acetyl groups in sugars A and E of chromomycin contribute distinctively in the DNA complex formation by providing additional H bond acceptor groups that interact with the 2-amino groups of G-bases, thus adding more specificity to DNA binding (6). Also, it has been recently demonstrated that elimination of acetyl groups leads to the generation of a chromomycin derivative with a significant decreased antitumor activity (14).We have recently reported the cloning and characterization of the chromomycin A 3 biosynthetic gene cluster (15). Because mithramycin and chromomycin share the same aglycone but differ in four of the five sugars attached to the aglycone, we were interested in characterizing the chromomycin glycosyltransferase genes. These genes together with those of the mithramycin cluster may serve as genetic tools for further studies on the generation of novel aureolic acid group derivatives with potential antitumor activity. Here we report the identification * Corresponding author. Mailing address for Carmen Méndez (molecular biological questions):