285 Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms Ben Shen Recent literature on polyketide biosynthesis suggests that Three types of bacterial PKSs are known to date. First, polyketide synthases have much greater diversity in both type I PKSs are multifunctional enzymes that are orga- mechanism and structure than the current type I, II and III nized into modules, each of which harbors a set of distinct, paradigms. These examples serve as an inspiration for searching non-iteratively acting activities responsible for the cata- novel polyketide synthases to give new insights into polyketide lysis of one cycle of polyketide chain elongation, as biosynthesis and to provide new opportunities for combinatorial exemplified by the 6-deoxyerythromycin B synthase biosynthesis. (DEBS) for the biosynthesis of reduced polyketides (i.e. macrolides, polyethers and polyene) such as erythro- Addresses mycin A (1)(Figure 1a) [1]. Second, type II PKSs are Division of Pharmaceutical Sciences and Department of Chemistry, multienzyme complexes that carry a single set of iteratively University of Wisconsin, Madison, WI 53705, USA acting activities, as exemplified by the tetracenomycin e-mail: [email protected] PKS for the biosynthesis of aromatic polyketides (often polycyclic) such as tetracenomycin C (2)(Figure 1b) [2]. Current Opinion in Chemical Biology 2003, 7:285–295 Third, type III PKSs, also known as chalcone synthase- like PKSs, are homodimeric enzymes that essentially are This review comes from a themed section on iteratively acting condensing enzymes, as exemplified by Biocatalysis and biotransformation Edited by Tadhg Begley and Ming-Daw Tsai the RppA synthase for the biosynthesis of aromatic poly- ketides (often monocyclic or bicyclic), such as flavolin (3) 1367-5931/03/$ – see front matter (Figure 1c) [3]. Type I and II PKSs use acyl carrier protein ß 2003 Elsevier Science Ltd. All rights reserved. (ACP) to activate the acyl CoA substrates and to channel DOI 10.1016/S1367-5931(03)00020-6 the growing polyketide intermediates, whereas type III PKSs, independent of ACP, act directly on the acyl CoA substrates. Despite structural and mechanistic differ- Abbreviations ACP acyl carrier protein ences, all types of PKSs biosynthesize polyketides by AT acyl transferase sequential decarboxylative condensation of the acyl CoA DEBS 6-deoxyerythromycin B synthase precursors, and the ketoacyl synthase (KS) domain (for DH dehydratase type I PKSs) or subunit (for type II and III PKSs) KR ketoreductase KS ketoacyl synthase catalyzes the C–C bond-forming step. NRPS nonribosomal peptide synthetase PKS polyketide synthase Since the first reports of bacterial type I PKS in 1990 [4,5], TD terminal domain type II PKS in 1984 [6,7], and type III PKS in 1999 [8], the PKS paradigms have served the scientific community beyond the call of duty, providing the molecular basis Introduction to explain the vast structural diversity observed with Polyketides are a large family of natural products found in polyketide natural products, and the biotechnological bacteria, fungi and plants, and include many clinically platform to produce ‘unnatural’ natural products by com- important drugs such as tetracycline, daunorubicin, ery- binatorial biosynthesis methods with engineered PKSs. thromycin, rapamycin and lovastatin. They are biosynthe- As the field stumbles into its adolescence, how much do sized from acyl CoA precursors by polyketide synthases we really know about polyketide biosynthesis? Here, (PKSs). Much of the current research on polyketide selected examples from recent literature are presented biosynthesis is driven by: first, the unparalleled biological to argue that PKSs have much greater diversity in both activities and enormous commercial value of these natural mechanism and structure than the currently well appre- products, which remain the most successful candidates for ciated type I, II and III paradigms. These examples serve new drug discovery; second, the extraordinary structure, as an inspiration in searching for novel PKSs, both to give mechanism and catalytic reactivity of PKSs that provide new insights into polyketide biosynthesis and to provide an unprecedented opportunity to investigate the mole- new opportunities for combinatorial biosynthesis. cular mechanisms of enzyme catalysis, molecular recog- nition and protein–protein interaction; and third, the Iterative type I PKSs for aromatic polyketide remarkable versatility and amenability of PKSs that allow biosynthesis the generation of novel compounds, difficult to access by Although aromatic polyketide biosyntheses in fungi are other means, by combinatorial biosynthesis methods. catalyzed by iterative type I PKSs, as exemplified by the www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:285–295 286 Biocatalysis and biotransformation Figure 1 (a) Type I PKS (noniterative) Loading PKS PKS PKS PKS (module-1) (module-2) (module-3) (module-4) (module-4) AT ACP KS AT KR ACP KS AT ACP KS AT KR DH ACP KS AT KR DH ACP S S S S O O O O S R R O O O OH O O O O O O O O CoAS OH DEBS OH OH + N OO HO HO − CoA (7 x) O OH O O O CoAS O − CO2 (6 x) OOH OOOCH3 (10 x) OH O 1 (b) Type II PKS (iterative) KSα KSβ ACP KSα KSβ ACP x y x y S S SH S O O O R z O O O R O O O OH O OO O O HO OH OH TcmKLM H3CCHOO3 CoAS O S-Enz OH OCH3 (10 x) − O − CO CoA OOOCHO 2 OH OH OH CH O OH 3 − 3 O O OH CH O − CO2 H2O 3 (10 x) (4 x) 2 (c) Type III PKS (ACP-independent & iterative) CoA CoA KS KS n n S S S O SH O O m O O O O O O S-Enz O OH OH OH O O OO RppA O CoAS O O O HO OH HO OH − O − CO (5 x) CoA (5 x) 2 O − − CO2 (4 x) H2O 3 Current Opinion in Chemical Biology Current Opinion in Chemical Biology 2003, 7:285–295 www.current-opinion.com Polyketide biosynthesis beyond the current paradigms Shen 287 6-methylsalicyclic acid synthase, the paradigm for aro- finding of NcsB from the neocarzinostatin (7) biosyn- matic polyketide biosyntheses in bacteria is the iterative thetic gene cluster suggests that bacterial iterative type I type II PKS (Figure 1b) [2]. However, because most of PKSs are not limited to monocyclic aromatic polyketide the type II PKSs studied so far were cloned according to biosynthesis and could catalyse the biosynthesis of the type II PKS paradigm, caution has to be taken to higher-order aromatic polyketides, such as the naphtha- generalize type II PKSs for aromatic polyketide biosynth- linic acid moiety of 7 (hexaketide) (Figure 2a). In fact, esis in bacteria. Bechthold and co-workers cloned the first early attempts to clone the ncs biosynthetic gene cluster iterative type I PKS, AviM, for aromatic polyketide for 7 on the assumption that its naphthalinic acid moiety is biosynthesis in bacteria in 1997 [9]. The aviM gene biosynthesized by a type II PKS failed, and, retrospec- was discovered from the avilamycin (4; Figure 2a) bio- tively, this approach cannot be successful because the ncs synthetic gene cluster that was cloned from Streptomyces cluster harbors no type II PKS gene. We succeeded in viridochromogens Tu¨ 57 using a deoxysugar biosynthetic cloning the ncs gene cluster by chromosomal walking from gene as a probe. AviM has the characteristic type I PKS the ncsA gene that encodes the neocarzinostatin apo- domains of KS, acyl transferase (AT), dehydratatse (DH), protein [12]. Two PKS genes were identified within and ACP (Figure 2b). Because the orsellinic acid moiety the ncs gene cluster. One of them, ncsB, encodes a type I is the only structural element in 4 that could be of PKS consisting of the characteristic KS, AT, DH, ketor- polyketide origin (Figure 2a), Bechthold and co-workers eductase (KR) and ACP domains (Figure 2d). In a expressed aviM in Streptomyces lividans TK24 and Strep- mechanistic analogy to AviM and CalO5, NcsB could tomyces coelicolour CH999 to verify if AviM can catalyzes be envisaged as catalysing the biosynthesis of naphtha- orsellinic acid (5) biosynthesis in vivo. Production of 5 was linic acid (8) from the acyl CoA precursors in an iterative observed in both hosts, confirming AviM as an orsellinic process, with an exception of regiospecific reduction at acid synthase that catalyzes aromatic polyketide bio- C-6 by the KR domain (Figure 2e). This hypothesis is synthesis from the acyl CoA precursors in an iterative consistent with the remarkable homology in both amino process (Figure 2c). Ironically, AviM was treated more as acid sequence and domain organization among NcsB, an exception to the type II PKS paradigm than as an AviM and CalO5. NcsB, therefore, represents the third indication that aromatic polyketide biosynthesis in bac- example of bacterial iterative type I PKSs for aromatic teria could be catalyzed by iterative type I PKS. polyketide biosynthesis. Thorson and co-workers [10] recently cloned the cali- Iterative type I PKSs for enediyne cheamicin (6) biosynthetic gene cluster from Micromonos- biosynthesis pora echinospora ssp. calichensis by screening for genes The enediyne family of antibiotics is structurally char- conferring 6 resistance and subsequently sequenced acterized by the enediyne core, a unit consisting of two and characterized the cal cluster [11]. Two PKS genes, acetylenic groups conjugated to a double bond or inci- calE8 and calO5, were identified within the cluster, each pient double bond within a nine-membered ring (e.g. 7 of which is characteristic of type I PKS. There are two and C-1027 (9)) or ten-membered ring (e.g. 6)(Figure 2a). structural elements within 6 that are of polyketide origin: Although feeding experiments with 13C-labeled precur- the enediyne core and the orsellinic acid moiety sors unambiguously established that both the nine- and (Figure 2a).