Engineering a Methymycin/Pikromycin−Calicheamicin Hybrid:  Construction of Two New Macrolides Carrying a Designed Sugar Moiety

Abstract

Nature continues to be the inspiration for most pharmaceutical drug leads, and given the synthetic challenge posed by many complex secondary metabolites, the emerging field of combinatorial biosynthesis has become a rich new source for modified non-natural scaffolds. 1 Yet, many naturally occurring bioactive secondary metabolites possess unusual carbohydrate ligands, which serve as molecular recognition elements critical for biological activity. 2 Without these essential sugar attachments, the biological activities of most clinically important secondary metabolites are either completely abolished or dramatically decreased. We and others have demonstrated that glycosyltransferases, responsible for the final glycosylation of certain secondary metabolites, show a high degree of promiscuity toward the nucleotide sugar donor. 3,4 These discoveries have opened the door to the possibility of manipulating the corresponding biosynthetic pathways for modifying the crucial glycosylation pattern of natural, or non-natural, secondary metabolite scaffolds in a combinatorial fashion. To date, the genetic manipulation of the carbohydrate appendage for any given metabolite has been limited to alterations and/or knock-outs of the small subset of genes required to construct and attach each desired carbohydrate moiety. However, a significant expansion of the saccharide structure diversity obtained by these methods might be accomplished via the recruitment and collaborative action of sugar genes from a variety of biosynthetic pathways to construct composite clusters with the potential to make and attach non-natural sugars.To test this possibility, we selected the Streptomyces Venezuelae methymycin/pikromycin gene cluster as the parent system and a gene from the calicheamicin biosynthetic gene cluster (from Micromonospora echinospora spp. Calichensis) as the foreign collaborator gene. The parent cluster encodes the biosynthetic enzymes for methymycin (1), neomethymycin (2), pikromycin (3), and narbomycin (4), all of which are macrolides containing desosamine (5) as the sole sugar component crucial for antibiotic activity. 5 Eight open reading frames (desI-desVIII) in this cluster have been assigned as genes involved in desosamine biosynthesis (Scheme 1). The antitumor agent calicheamicin (6) contains four unique sugars crucial to tight DNA binding (K a ≈ 10 6 -10 8 ), one of which (9) is derived from 4-amino-4,6-dideoxyglucose (8) and is part of the unusually restricted N-O connection between sugars A and B (Scheme 2). 6 Compound 8 is anticipated to be derived from the corresponding 4-ketosugar 7 via a transamination reaction, and recent work has led to the assignment of a gene (calH) as encoding the desired C-4 aminotransferase (Scheme 2). 7 Interestingly, the proposed substrate for CalH, 7, is also an intermediate in the desosamine pathway and is expected to exist Thamchaipenet, A.; Gustafsson, C.; Fu, H.; Betlach, M.; Betlach, M.; Ashley, G. Targeted deletion/disruption of the desVI, desV, or desI genes in the methymycin/pikromycin...

Biosynthesis and Combinatorial Biosynthesis of Pikromycin-Related Macrolides in Streptomyces venezuelae

“…A more elaborate experiment by Zhao et al (1999) involved combining the pikromycin pathway and a sugar biosynthetic gene, calH, from the calicheamicin biosynthetic pathway from Micromonospora echinospora spp. calichensis using the newly constructed pikAI promoter-driven host vector system.…”

A New Role for Polyketides

“…More and more new polyketides are created through the developing advanced techniques of combinatorial biosynthesis, [2] and new directions can be observed, such as attempts to link the biosyntheses of modular polyketides and nonribosomal peptides [3] and the attachment of important deoxysugar moieties to various polyketide-derived aglycones. [4] Recently, a previously totally unknown role of polyketides has been discovered by Small and co-workers, in the laboratories of the National Institutes of Health in Hamilton, Montana, USA; this is in the role of a toxin which is required for virulence in context with Buruli ulcers caused by Mycobacterium ulcerans. [5] The latter is a chronic and progressive necrotizing ulcer for which no medical treatment exists.…”

“…[3] This hypothesis was recently strengthened by the findings of Snyders group at the Johns Hopkins University in Baltimore. [4] They succeeded in cloning and sequencing a serine racemase from a mammalian brain. This protein represents a novel enzyme, possibly a first member of a whole family of enzymes, without any significant homology to other known amino acid racemases from lower organisms (with the exception of a short consensus sequence forming the binding site for pyridoxal phosphate).…”

The Structural Basis for Substrate Anchoring, Active Site Selectivity, and Product Formation by P450 PikC from Streptomyces venezuelae

“…The Pik 3 biosynthetic gene cluster of Streptomyces venezuelae represents an effective system for the synthesis of novel polyketide antibiotics due to (i) its ability to generate two macrolactone ring systems; (ii) the presence of a flexible desosaminyl transferase (DesVII) that is tolerant of changes in macrolactone structure (13,18,19), as well as modifications to the sugar substituent (14)(15)(16)(17); and finally, (iii) the unusual P450 monooxygenase (CYP) PikC (CYP107L1), responsible for the diverse pattern of hydroxylated natural products obtained from the Pik pathway (Fig. 1).…”