Commentary Combinatorial biosynthesis: Lesson learned from nature

Polyketides produced by bacteria and fungi represent an extremely rich source of biologically active compounds that find wide-ranging applications as antibiotics, immunosuppresants, antitumor agents, and antiparasitic agents. The striking structural diversity of polyketides that gives rise to these wide-ranging applications is dictated both by the polyketide synthase (PKS) responsible for assembling the carbon backbone of the molecule, and by the tailoring enzymes (such as glycosyl transferases and hydroxylases) that modify the polyketide ring. Seminal work in our understanding of the complex polyketides came a number of years ago with the revelation that erythromycin produced by Saccharopolyspora erythraea is assembled by a modular PKS (1, 2). The first module is responsible for priming erythromycin with propionyl CoA and catalyzing a decarboxylative condensation with methylmalonyl CoA. The remaining five modules catalyze successive condensations with methylmalonyl CoA before a terminal thioesterase (TE) domain releases the aglycone, deoxyerythronolide B. The presence or absence of ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) catalytic domains within the module controls the extent to which the b-carbonyl of the growing polyketide chain is processed. In this issue of the Proceedings researchers at the University of Minnesota have revealed that a similar modular PKS is responsible for ketolide biosynthesis in Streptomyces venezulae (Fig. 1) (3). The discovery of the modular polyketide synthase has prompted two questions to be addressed: how is structural diversity achieved by using a modular type I PKS and can such a PKS be manipulated to access additional structural diversity? Recent analysis of additional modular PKS systems, including those involved in rapamycin, rifamycin, and niddamycin biosynthesis, has shown that a PKS polypeptide can contain 1–6 modules (4–6). The total number of modules controls the length of the polyketide chain that is formed before cyclization, whereas the catalytic domains of each module control the level of oxidation along the polyketide chain. The specificity of the acyl transferase domain within each module, which specifies for a malonyl CoA, methylmalonyl CoA, or ethylmalonyl CoA extender unit, controls the level of branching along polyketide chain (7, 8). Additional structural diversity is accomplished by the loading domains in the first module, which use a wide range of starter units such as 3-amino-5-hydroxybenzoic acid, isobutyryl CoA, and 3,4-dihydroxycyclohexanecarboxylic acid (4, 5, 9). Enzymes that catalyze the cyclization of the polyketide product also can contribute significantly to the structural diversity (10). In addition to these studies of natural PKSs, hybrid polyketides have been generated by a variety of techniques. Ring-contracted polyketides have been produced by movement of the terminal thioesterase to different modules of the PKS (11, 12), whereas analogues of the parent polyketide product have been produced by deletions, switches, and insertions of various catalytic domains and modules (13–16). To date these studies have demonstrated that a single hybrid or natural polyketide synthase system typically is responsible for formation of a single product (thus S. hygroscopicus, which produces at least four complex polyketides, contains four separate PKS gene clusters; ref. 17). In addition, it has been shown that hybrid polyketide products are not always effective substrates for post-polyketide tailoring enzymes, as these often exhibit a strict substrate specificity (1, 15). In this issue of the Proceedings, Xue et al. (3) report a unique observation that a single PKS within a cluster of biosynthetic genes in S. venezulae is responsible for formation of both 12and 14-membered macrolactones (3). In addition, the researchers have determined that two of the postpolyketide modifying enzymes have an unusually broad substrate and regiochemical specificity. The first significant finding by Xue et al. is that four polypeptides, PikAI, PikAII, PikAIII and PikAIV, are involved in the production of the 14-membered ring macrolactone, narbonolide (the precursor to narbomycin and pikromycin), whereas just PikAI, PikAII, and PikAIII are required for production of the 12-membered macrolactone ring 10deoxymethynolide (a precursor to methymycin and neomethymycin) (Fig. 1). In the pikromycin PKS the last two modules (modules 5 and 6) are encoded by separate ORFs, whereas previously identified PKS clusters that produce 14-membered macrolides contain both of these within a single ORF (1, 18). The researchers suggest that it is this feature that allows for early termination of the polyketide chain. Xue et al. were faced with an intriguing question regarding the manner in which the nascent polyketide product is released from PikAIII and cyclized to give the 12-membered macrolide because, unlike PikAIV, this polypeptide does not contain a terminal TE domain. A hypothesis that a type II monofunctional thioesterase (Pik TEII encoded by pikAV) plays an important role was supported by the demonstration that inactivation of the corresponding pikAV gene led to a mutant that generated less than 5% of the 12-membered macrolide products. Xue et al. point out that various engineered polyketides of different chain lengths have been made by repositioning of the TE catalytic domain to different modules on a PKS (11, 12, 19), but that each positioning of the TE leads to only one discrete polyketide chain length product. The researchers raise an intriguing possibility that TEIIs along with novel PKS systems may allow for the simultaneous production of polyketides of different chain lengths. It should be noted, however, that many natural polyketide and nonribosomal polypeptide biosynthetic clusters (including the erythromycin PKS gene cluster) already contain homologues of Pik TEII (20), yet under most conditions produce predominantly one macrolide product. The utility of a Pik TEII in combinatorial biology technologies clearly will require further investigation. Xue et al. also discovered that 5% of the narbonolidederived polyketides were produced in the pikAV mutant and argue that Pik TEII plays a role in release of the nascent