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Combinatorial Biosynthesis of Reduced Polyketides REVIEWS COMBINATORIAL BIOSYNTHESIS OF REDUCED POLYKETIDES Kira J. Weissman and Peter F. Leadlay Abstract | The bacterial multienzyme polyketide synthases (PKSs) produce a diverse array of products that have been developed into medicines, including antibiotics and anticancer agents. The modular genetic architecture of these PKSs suggests that it might be possible to engineer the enzymes to produce novel drug candidates, a strategy known as ‘combinatorial biosynthesis’. So far, directed engineering of modular PKSs has resulted in the production of more than 200 new polyketides, but key challenges remain before the potential of combinatorial biosynthesis can be fully realized. 3,4 SCAFFOLD Polyketide natural products are among the most polyketide antibiotic erythromycin A . By mixing The backbone atoms of a important microbial metabolites in human medicine, and matching some of the actinorhodin PKS genes molecule, on which targeting both acute and degenerative diseases. They with their counterparts from other aromatic-antibiotic functionality is displayed. are in clinical use as antibiotics (erythromycin A, gene clusters, hybrid molecules were often produced5,6, rifamycin S), anticancer drugs (doxorubicin, epothi- giving rise to the concept now known as ‘combinatorial lone), cholesterol-lowering agents (lovastatin), anti- biosynthesis’7. Given the modular organization within parasitics (avermectin), antifungals (amphotericin B), the giant PKS proteins that catalyse erythro mycin insecticides (spinosyn A) and immunosuppressants biosynthesis, simple mixing and matching of these (rapamycin) (FIG. 1). Polyketide-derived pharma- intact proteins would be of limited interest. However, ceuticals comprise 20% of the top-selling drugs, with once methods were devised to ‘cut and paste’ portions combined worldwide revenues of over UK£10 billion of these giant genes to produce chimaeric genes, in a per year. way that retained the activity of the multiple enzyme There is intense interest in discovering analogues activities that they encode, it became apparent that the of polyketide natural products with improved or structure of erythromycin (and therefore other related novel pharmacological properties — the need for polyketides) might be rationally modified by creating new anti biotics is particularly well recognized1. patchwork PKS genes from two or more natural PKS Polyketide-drug discovery efforts have traditionally genes spliced together8–10, an idea which now dominates taken two forms: isolation and identification of new combinatorial biosynthesis11–13. compounds from the biosphere, or the semi-synthetic Although modular PKS genetic engineering has modification of existing drug SCAFFOLDS using medici- now repeatedly been demonstrated14–16, the low effi- nal chemistry. Both approaches are likely to yield ciency of these experiments limits their routine use in new structures in the foreseeable future. For the past industrial drug discovery, and this type of combina- Department of few years, however, an alternative strategy has also torial engineering remains too labour intensive to be Biochemistry, University of been used to produce new polyketides — genetically described as high-throughput. Therefore, research has Cambridge, Cambridge, engineering polyketide synthase (PKS) biosynthetic also focused on developing improved tools for genetic CB2 1GA, UK. pathways. This advance was made possible by the engineering and heterologous expression, as well as e-mails: [email protected]; initial cloning of several polyketide gene clusters in understanding the structure and function of modular [email protected] the 1980s, including the clusters for synthesis of the PKS proteins and post-PKS tailoring enzymes. These doi:10.1038/nrmicro1287 aromatic antibiotic actino rhodin2, and the reduced efforts have recently resulted in a biological source of NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | DECEMBER 2005 | 925 © 2005 Nature Publishing Group REVIEWS a Reduced polyketides O OCH3 O HO O H3CO OH OH OH O H3CO O HO O O OH O O O O O NH H3CO O O N O H3CO O O OH HOOC O O Rapamycin O Monensin A OH Rifamycin S Polyketide-peptide Polyether antibiotic Ansamycin antibiotic immunosuppressant OCH3 HO O OCH3 O O O O HO O O OH OH O OH O O OO HOOOH OH OH O OH COOH O O O O OH OH NH2 Amphotericin B OH Lovastatin Avermectin B1b Polyene antifungal Cholesterol-lowering agent Macrolide antiparasitic O HO OH (H3C)2N O O N(CH ) OCH3 S OH 3 2 O OCH HO O 3 HO OCH3 N O O O O O O O O OCH3 O O OH O O OH Epothilone Polyketide-polypeptide Erythromycin A Spinosyn A anticancer agent Macrolide antibiotic Macrolide insecticide b Aromatic polyketides c Unclassified O OCH3 O OH O OH O N H OH O O HOOC O O O O COOH OCH O OH O 3 OH OH O O O O (H3C)2N OH OH NH2 OH OH O O O O HO OH Doxorubicin Aromatic antitumour Actinorhodin C-1027 Cl agent Aromatic antibiotic Enediyne antitumour agent NH2 Figure 1 | Classes of polyketide metabolites and representative structures. This conventional classification distinguishes those metabolites (a) in which polyketide-chain synthesis occurs with concomitant reduction from (largely) aromatic metabolites, such as in (b). However, many polyketides (c) resist easy classification. 926 | DECEMBER 2005 | VOLUME 3 www.nature.com/reviews/micro © 2005 Nature Publishing Group REVIEWS the semi-synthetic drug ivermectin14, as well as several bacteria24. The actinomycetes, a diverse order of promising chemotherapeutic-drug leads derived from Gram-positive bacteria, synthesize most of all known geldanamycin15 and epothilone16. polyketides. Among them, the soil-dwelling, filamen- tous Streptomyces and Saccharopolyspora species are Polyketides: structure and function the most prolific polyketide producers, although myxo- Polyketides make good drugs. Of the 7,000 known bacteria25 and pseudomonads26 are also important polyketide structures, more than 20 (0.3%) have been sources of these compounds. commercialized, which is considerably better than The functions of polyketides, which are non- the typical <0.001% ‘hit rate’ from standard pharma- essential for the growth of producer organisms at least ceutical screens. Despite their relatively low molecu- in vitro, are unknown27. It is often assumed that they lar weights (on average 800 Da), polyketides bristle provide a competitive advantage in the environment with diverse functional groups, and many are rich in through uses in intra- and interspecies communication stereochemistry. or in self-defence. Self-defence as an explanation is par- Polyketides have been classified both on the basis ticularly persuasive for non-motile Streptomyces spp.27 of their structures and on their mode of biosynthesis. Others have argued that some secondary metabolites For simplicity, we distinguish between polyketides have no value at present to their producer organisms, that are derived from unreduced polyketone chains but that the ability to produce these molecules provides and are largely aromatic (for example, doxorubicin a selective advantage28. (FIG. 1b)) and those in which many of the carbonyl functionalities have been reduced during bio synthesis PKSs are modular (for example, erythromycin A (FIG. 1a)). In the case For the past 15 years, the biosynthesis of the polyketide of the reduced polyketides, the initial linear chain core of erythromycin A, 6-deoxyerythronolide B is often constrained by MACROCYCLIZATION into a (6-dEB) (FIG. 2), has provided the paradigm for under- biologically active conformation, which promotes standing the structure and function of the PKSs that precise interaction with protein targets17. In this are responsible for assembling complex polyketides. review, we will focus only on the reduced polyketides 6-dEB is constructed on a gigantic molecular ‘assem- produced by modular PKSs, as their biosynthesis has bly line’, with a dedicated enzymatic domain for each to date proved most amenable to rational genetic biosynthetic step29 (FIG. 2), a feature known as the engineering. ‘collinearity rule.’ Reduced polyketides can be divided into two The boundaries and active sites of the PKS main classes, the macrocycles and the polyethers. domains in the gene sequence were identified by Macrocycles, such as erythromycin A and rapamycin their homologies to the corresponding enzymes in (FIG. 1a), contain a large lactone or lactam ring18 and animal fatty-acid biosynthesis30. Sequence analysis have a wide variety of biological activities. Antibiotic revealed that the domains are covalently linked and and antifungal polyenes19, such as amphotericin B grouped into functional units called ‘modules’, so (FIG. 1a) and nystatin, form a subset of the macrolides that each module carries out a single chain-extension that incorporate three to seven conjugated double step and any required processing reactions before the bonds. The polyene macrolides are among the giants intermediate is passed on to the downstream module of the polyketide world, with rings that contain more (FIG. 2); therefore, these multienzyme complexes have than 44 carbons. A second subset of the macrolides is been named the modular PKSs. The modules are the ansamycin antibiotics, which includes rifamycin S further organized into multimodular subunits — in (FIG. 1a) and geldanamycin. These molecules incorporate erythromycin biosynthesis these subunits are 6-dEB the non-proteinogenic amino acid 3-amino-5-hydroxy- synthases (DEBS) 1, 2 and 3. A typical subunit con- benzoic acid (AHBA) as the first building block and, in tains two or three modules, although a multienzyme
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