Generation of Hybrid Elloramycin Analogs by Combinatorial Biosynthesis Using Genes from Anthracycline-Type and Macrolide Biosynthetic Pathways

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tu2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product. Search for new bioactive drugs has been usually carried out by pharmaceutical companies through wide screening programs for the isolation of microorganisms producing useful bioactive compounds. Succesful screening programs have produced most of the clinically useful drugs. In addition, chemical modification of selected compounds introducing specific changes in these lead compounds has also rendered important families of bioactive compounds, as it is the case of the different generations of s-lactam antibiotics. The development of recombinant DNA technology is beginning to offer another alternative for the generation of bioactive compounds through the genetic manipulation of biosynthetic pathways. Many bioactive compounds with pharmaceutical, veterinary or agricultural applications belong to the polyketide family. This is one of the largest families of secondary metabolites and includes well known bioactive compounds such as antibiotics (macrolides, tetracyclines), antitumor agents (anthracyclines), antiparasites (avermectins), immunosuppressant agents (rapamycin, FK506), etc. Polyketides show very diverse chemical structures. They all are synthesized in the early stages of their biosynthesis through the condensation of short-chain carboxylic acids in a series of reactions catalyzed by complex enzymes named polyketide synthases (Hopwood and Sherman, 1990; Katz and Donadio, 1993; Hutchinson and Fujii, 1995; Hopwood, 1997). An interesting feature of many polyketide drugs is the presence of sugars attached to the polyketidederived aglycones. All these sugars belong to the family of 6-deoxyhexoses, whose members are present in a great variety of secondary metabolites (Piepersberg, 1994; Liu and Thorson 1994; Kirschning et al., 1997; Trefzer et al., 1999). In many cases, the presence of the sugars is important or even essential for bioactivity (Kirschning et al., 1997). Therefore, engineering glycosylation in polyketide drugs could be a powerful tool for generating structural biodiversity that might conduct to the generation of novel bioactive drugs. Obviously this will require a certain degree of flexibility of glycosyltransferases. Elloramycin is an anthracycline drug structurally related to tetracenomycin C. They differ in the absence of a C12a-O methylation in tetracenomycin C (which is present in elloramycin) and the glycosylation of the elloramycin