- Email: [email protected]
Developing tools for engineering hybrid polyketide synthetic pathways
Developing tools for engineering hybrid polyketide synthetic pathways Jeffrey D Kittendorf and David H Sherman Bacterial type I polyketide synthases (PKSs) are complex, multifunctional enzymes that synthesize structurally diverse and medicinally important natural products. Given their modular organization, the manipulation of type I PKSs holds tremendous promise for the generation of novel compounds that are not easily accessible by standard synthetic chemical approaches. In theory, hybrid polyketide synthetic pathways can be constructed through the rational recombination of catalytic domains or modules from a variety of PKS systems; however, the general success of this strategy has been elusive, largely due to a poor understanding of the interactions between catalytic domains, as well as PKS modules. Over the past several years, a fundamental knowledge of these issues, and others, has begun to emerge, offering refined strategies for the facile engineering of hybrid polyketide pathways. Addresses University of Michigan Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, Microbiology & Immunology, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA Corresponding author: Sherman, David H ([email protected])
Current Opinion in Biotechnology 2006, 17:597–605 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 12th October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.09.005
Introduction Historically, nature has been a rich source of structurally complex organic molecules that have proven beneficial to human health [1]. Currently, there are many examples of natural product-derived pharmaceuticals that are employed to combat human disease [2], and those classified as polyketides are among the most important. Polyketides represent a large class of microbial and plant-derived metabolites that display a dazzling combination of structural diversity and pharmacological activity [3]. These organic molecules vary in molecular weight, functional group modification and complexity, and include linear, polycyclic and macrocyclic structural forms. Most remarkable, their medicinal impact spans a growing range of human and animal diseases. At present, polyketide-based pharmaceuticals find clinical utility as www.sciencedirect.com
antibiotics, antiparasitic agents, antifungals, anticancer drugs and immunosuppressants (Figure 1). Because of their therapeutic potential, an ever-increasing demand has been placed on current natural product research to uncover novel polyketide compounds. Although their discovery continues to proceed at a rapid pace, the clinical development of these compounds remains constrained by the fact that their isolation from natural hosts is often limited in scale. Furthermore, the structural complexity displayed by this class of compounds poses substantial challenges for the total synthesis of the target natural product and its analogs. These limitations have significantly hindered the efforts to modulate the desired combination of pharmacological, pharmacokinetic and pharmacodynamic properties of lead compounds. Recent efforts to increase both the yield and the structural diversity of polyketide natural products have focused on harnessing the catalytic power and specificity of the enzymes responsible for polyketide biosynthesis. The biosynthesis of macrolide polyketides relies on type I modular polyketide synthases (PKSs) to catalyze the stepwise condensation of simple carboxylic acid derivatives (e.g. malonyl-CoA and methylmalonyl-CoA). Organizationally, type I PKSs are arranged into modules, wherein each module is comprised of a set of catalytic activities that is responsible for a single elongation of the polyketide chain, and subsequent reductive processing of the b-keto functionality (Figure 2) [4]. The sequential arrangement of modules within a PKS system effectively serves as a biosynthetic assembly line, responsible for dictating the final size and structure of the polyketide scaffold. Readers interested in additional background information regarding PKS systems are referred to one of the many recent reviews that have been written on this topic [5–9]. The modular nature of type I PKSs offers several strategies for the generation of structurally diverse polyketides through a combinatorial ‘plug and play’ approach [10,11]. For example, the alteration of individual catalytic domains, via inactivation, substitution, addition or deletion, can yield predicted structural alterations of the final PKS product. Likewise, the addition, deletion or exchange of intact modules can also impart structural variety into polyketide metabolites. Using these and other approaches, over 200 novel structures have been generated. Although impressive, these successes appear to be more the exception rather than the rule, as many Current Opinion in Biotechnology 2006, 17:597–605
598 Chemical biotechnology
Figure 1
Examples of clinically important polyketides.
efforts result in trace levels, or fail to produce the desired product. A recent theoretical analysis suggests that modular PKSs have the potential to generate hundreds of millions of compounds through the novel assembly of catalytic units [12]; however, it has become increasingly apparent that before this potential can be met, much remains to be learned regarding the intricacies of domain and modular interactions within PKS systems. In this review, recent advances in the understanding of these issues are highlighted, and their application toward the construction of hybrid PKS systems is discussed.
Manipulation of PKS catalytic domains It is well established that the arrangement of catalytic domains within a PKS module directly influences polyketide structure [4]. As such, the manipulation of catalytic domains presents an attractive strategy for the generation of novel chemotypes. At a minimum, each PKS elongation module contains an acyl transferase (AT) Current Opinion in Biotechnology 2006, 17:597–605
domain, an acyl carrier protein (ACP) domain and a ketosynthase (KS) domain (Figure 2). The AT is responsible for specifying and loading the appropriate CoA extender unit onto the ACP (e.g. malonyl-CoA, methylmalonyl-CoA or methoxymalonyl-CoA). The KS domain then catalyzes a decarboxylative condensation of the extender unit with the growing polyketide chain obtained from the preceding module to generate an ACP-bound b-ketoacyl product. In addition to these three core domains, each elongation module can contain up to three additional domains that are responsible for reductive processing of the b-keto functionality before the next extension step. The presence of a ketoreductase (KR) domain gives rise to a b-hydroxyl functionality; the presence of both a KR and a dehydratase (DH) domain generates an alkene, whereas the combination of KR, DH and enoyl reductase (ER) results in complete reduction to an alkane. Finally, termination of polyketide biosynthesis is catalyzed by a thioesterase (TE) domain www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 599
Figure 2
The pikromycin PKS: an example of a modular polyketide synthase. The pikromycin PKS contains one loading module and six elongation modules spanning four polypeptide chains (PikAI–PikAIV). Chain elongation through PikAIII followed by TE-catalyzed termination results in the 12-membered macrolactone product, 10-deoxymethynolide, whereas continued elongation of the polyketide chain produces the 14-membered macrolactone, narbonolide. Both products undergo further processing via a glycosyl transferase and P450 hydroxylase to yield the methymycin and pikromycin suite of antibiotics. Abbreviations: ACP, acyl carrier protein; AT, acyl transferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KR0, inactive ketoreductase; KS, ketosynthase; KSQ, decarboxylase; TE, thioesterase.
located at the carboxy terminus of the final elongation module (Figure 2). Alteration of PKS catalytic domains can provide access to structural diversity that is not easily achieved via standard synthetic methodology. For example, genetic manipulation of AT domains can effectively modify the carbon substituents that decorate the polyketide scaffold. Typically, the specificity of an AT domain (e.g. methylmalonyl-CoA versus malonyl-CoA) can be altered via sitedirected mutagenesis [13,14]. Alternatively, inactivated ATs can be complemented via exogenous AT-like enzymes acting in trans. Khosla and co-workers [15] have demonstrated that the completely inactivated AT domain www.sciencedirect.com
from module 6 of the 6-deoxyerythronolide B (DEBS) PKS can be rescued with a heterologous malonylCoA:ACP transacylase (MAT) to efficiently generate 2-desmethyl-6-deoxyerythronolide B. AT domains can also be genetically swapped between PKS systems [16]; however, this often results in reduced catalytic efficiency of the hybrid multifunctional protein, a phenomenon that has been attributed to an overall structural perturbation of the module. Most recently, researchers at Kosan Biosciences [17] have reportedly replaced the AT domains in six of the seven geldanamycin PKS modules with a malonyl-specific AT from the rapamycin biosynthetic cluster. Remarkably, four of the six hybrid modules successfully produced the predicted geldanamycin Current Opinion in Biotechnology 2006, 17:597–605
600 Chemical biotechnology
analogs, albeit in much lower yield relative to geldanamycin in the producing strain. The use of inaccurate domain boundaries presumably destabilizes the modular architecture, resulting in the two nonfunctional hybrids.
it is expected that this newfound structural information can be leveraged into more successful strategies for the incorporation of functional reductive domains into hybrid PKS systems.
It has recently been reported that limited proteolysis of DEBS module 3 produces a stable peptide fragment consisting of a KS–AT di-domain [18]. Subsequently, KS–AT di-domains from DEBS modules 3 and 6 have been cloned and overexpressed [18,19]. When presented with a separate ACP domain, the recombinant KS–AT di-domains demonstrate the ability to catalyze condensation activity, suggesting that maintenance of the KS–AT interface is crucial in the design of hybrid PKS modules. An enhanced understanding of this interaction has recently been gained from the crystal structure of the recombinant KS–AT di-domain from DEBS module 5 [20]. On the basis of this structure, it appears that the relative positions of the KS and AT domains are rigidly fixed via interactions with both the interdomain linker region and the linker region immediately downstream of the AT. This suggests that in domain exchange experiments, the accurate dissection and insertion of the AT is likely crucial for the maintenance of a strict spatial organization between KS and AT. Thus, structural disruption might be best minimized in a hybrid module via simultaneous swapping of native KS–AT di-domain pairs to preserve the natural interface; however, this approach remains to be experimentally explored.
DNA redesign: increasing the modularity of the PKS module
The incorporation of reductive processing domains into PKS modules can also lead to the generation of novel chemotypes [21]. As with the exchange of AT domains, the successful addition of reductive domains into PKS modules often results in attenuated catalytic efficiency. Currently, the lack of available structural information of PKSs precludes definitive identification of domain boundaries, thus presenting a major barrier for successful construction of hybrid PKS systems. Crystal structures of two fatty acid synthases (FASs, mammalian and fungal) have recently been reported [22,23]. Although the similarity that exists between FASs and PKSs offers hope that a structure for a PKS module is forthcoming, current efforts are focused on determining the structures of individual PKS domains. In addition to the previously described structure of the DEBS module 5 KS–AT di-domain [20], the structures of the TE domain from the DEBS PKS and pikromycin (Pik) PKS have been solved [24,25]. Most recently, Stroud and co-workers [26] have reported the structure of the KR domain (in the presence of an NADPH cofactor) from module 1 of the DEBS PKS, providing important insights into key structural details. From these data, the domain boundaries of the KR domain are readily defined. Moreover, an enhanced understanding of the domain interfaces that exist between all three reductive domains relative to ACP is provided. As such, Current Opinion in Biotechnology 2006, 17:597–605
The high percentage of G+C content, which is often characteristic of actinomycetes-derived PKS-encoding genes, makes genetic manipulation via standard molecular biology a challenging endeavor. To overcome this issue, a strategy has been developed for the efficient synthesis of long, contiguous fragments of DNA [27]. The appeal of this semi-synthetic technology is twofold. First, it allows for the reprogramming of the codon usage of a given PKS gene to one that is more amenable to gene expression in heterologous hosts, such as Escherichia coli. Second, it enables the incorporation of rationally positioned restriction enzyme recognition sequences, making downstream genetic exchange a much less complicated process. This technology relies on the chemical synthesis of 500 bp DNA ‘synthons’ that are subsequently assembled into 5 kb ‘multisynthons’ via the ligation by selection (LBS) method [28]. As proof of concept, the complete DEBS-encoding gene cluster has been redesigned, synthesized and expressed in E. coli [29]. The streamlined redesign and synthesis of large DNA fragments offers a general approach for the construction of hybrid PKS modules. Menzella et al. [30] have recently described a powerful strategy in which synthetic building blocks (i.e. catalytic domains or modules) from a variety of PKS systems can be synthetically engineered to incorporate unique restriction sites flanking either end. These engineered sites enable the facile plug and play recombination of catalytic domains and modules, a technique that has been referred to as the ‘Lego-ization’ of polyketide biosynthesis [31]. The general applicability of this approach was demonstrated by synthesizing 14 different modules from eight different PKS systems and rearranging them into 154 different bi-modular combinations, suitable for E. coli expression. Notably, just under half of the bi-modules generated the predicted triketide lactone product. However, each of the recombined modules demonstrated catalytic competence by processing unnatural substrates. This suggests that the lack of activity displayed by some bi-module combinations is most likely due to faulty communication between the upstream ACP and the downstream KS of interacting modules. Indeed, Santi and co-workers [32] have recently described the activation of nonproductive bimodular hybrids through KS domain exchange, whereby the KS domain of the acceptor module was replaced with the KS domain that naturally interacts with the upstream ACP. This success underscores the importance of optimizing modular interactions in the future design of hybrid PKS pathways. www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 601
Module exchange Perhaps the most obvious strategy for the construction of hybrid polyketide synthetic pathways is via de novo assembly of individual modules from a variety of PKS systems. The appeal of this approach lies in the fact that the enzymatic domains within a given PKS module have already been optimized to work as a contiguous unit. As such, structural perturbations that translate into catalytic inefficiency should be minimized. Chemical diversity can be implemented into the polyketide scaffold by assembling modules that contain the desired extender unit specificity and a combination of reductive domains without concern for optimizing domain interactions within any given module. However, the feasibility of this approach requires (i) efficient and specific communication between interacting ‘heterologous’ modules to facilitate channeling of the polyketide intermediates and (ii) broad substrate tolerance of the individual domains that comprise the heterologous system. Failure to meet these requirements would inevitably result in a significant barrier for the production of novel polyketides via hybrid PKS systems. The assembly-line mechanism employed by modular PKSs relies on the efficient transfer of linear intermediates between consecutive elongation modules. Specifically, the ACP-bound intermediate of the upstream module must be channeled to the downstream KS domain before initiating another round of chain elongation. For modules contained on a single PKS polypeptide (e.g. bi-, tri- and tetra-modular PKSs), this interaction appears to be facilitated by short peptide linkers between consecutive ACP and KS domains [33]. By contrast, interactions between modules contained on separate polypeptides are mediated via longer peptide domains, termed ‘docking domains’, that are present at the C-terminus and N-terminus of the interacting polypeptides [33]. A similar type of communication occurs between interacting modules of non-ribosomal peptide synthases (NRPSs), offering the possibility to engineer novel PKS–NRPS hybrid pathways [34]. The inherent specificity that exists between PKS docking domain partners has previously been established [35]. Recent work has begun to elucidate the structural basis for this molecular recognition. Utilizing protein NMR spectroscopy, the structure of the docking domain interaction that exists between DEBS module 4 and DEBS module 5 was determined [36]. This structure demonstrates that the C-terminal docking domain of DEBS module 4 is comprised of three separate helical regions, whereas the N-terminal docking domain of DEBS module 5 consists of a single helix that adopts a coiled-coiled structure within the dimeric PKS. However, it appears that the interaction between these two docking domains is mediated solely by the terminal helix of the C-terminal docking domain (two per dimer) of the ACP and the www.sciencedirect.com
coiled-coiled dimeric structure of the N-terminal docking domain of the KS, essentially forming an intermolecular four-a-helix bundle (Figure 3). A survey of sequences from a variety of interacting PKS modules suggests that this structural motif is a universal feature of PKS docking domains [36]. The current model of docking domain interactions between PKS modules suggests that, as an alternative to the complementary exchanging of entire docking domains [37], communication between heterologous PKS modules could be achieved by simply installing a matched pair of the interacting helices that participate in the four-a-helix bundle. To further examine this, a series of experiments has been described in which the putative docking helices from interacting docking domains were exchanged, in either matched, mismatched or covalently linked pairs [38]. The substitution of these helices with a matched pair from another set of interacting modules did not yield a significant difference in product formation, whereas substitution with matched, covalently linked pairs gave a slight increase in product yields. As expected, the substitution with mismatched pairs resulted in decreased product yield. It has been proposed that specific interactions between individual amino acids (referred to as code residues) within the docking helices might serve to stabilize matched partners [38]. For example, the interaction between the putative code residues of the matched docking pairs of DEBS 2 and DEBS 3 is attractive (Arg and Asp), whereas in the mismatched pairs of DEBS 1 and DEBS 3, the interaction between these code residues is repulsive (Glu and Asp), likely contributing to their destabilization. Further experimentation is required to fully explore this idea; however, if the role of the code residues is found to be general, it might enable facile changes to interactions between PKS modules by simply changing a few amino acid residues.
Generation of hybrid PKSs via gene shuffling Gene shuffling techniques are routinely used in directed enzyme evolution experiments to impart specific properties and activities into an enzyme of interest; however, until recently, these techniques have never been applied to the construction of hybrid PKS systems. Using the Pik PKS system (Figure 2), Reynolds and coworkers [39] have described a method that utilizes in vivo recombination with a counter selectable marker, followed by functional complementation in Streptomyces venezuelae to generate a library of hybrid PikAI modules. A bioassay screen of over 4000 transformants identified three functional hybrids, underscoring the innate challenges in successful construction of hybrid PKS modules. Nonetheless, the application of this method to a variety of PKS modules lends itself as a powerful tool for the functional recombination of PKS domains and modules. Current Opinion in Biotechnology 2006, 17:597–605
602 Chemical biotechnology
Figure 3
Model of PKS docking domains. A model highlighting the four-a-helix bundle involved in mediating the protein–protein interaction between DEBS module 4 and DEBS module 5. Shown in blue are the terminal helices from C-terminal docking domain of module 4 and in green is the N-terminal docking domain of module 5. The putative ‘code residues’ (arginine and aspartate) that have been postulated to provide electrostatic stabilization to the docking interaction are highlighted. This figure was generated from the PDB coordinates 1PZR [36].
Polyketide diversity via chemoenzymatic synthesis: an alternative to hybrid PKS systems In theory, the construction of hybrid PKS systems offers a tremendous opportunity to access difficult-to-synthesize chemical entities via rational genetic manipulation [12]. Despite recent successes, the ability to achieve a productive outcome from any single experiment involving rational mixing and matching of components from a variety of PKS systems remains uncertain. As an alternative to hybrid PKSs, recent efforts have begun to focus on the development of chemoenzymatic strategies for the generation of new polyketide chemical diversity. Within this approach, structural diversity can be incorporated during the chemical synthesis of analogs based on advanced polyketide chain-elongation intermediates. These molecules are subsequently incorporated as substrates to either individual PKS modules (for elongation and processing) or individual PKS domains (e.g. thioesterase) to generate the final polyketide product. Several studies using excised TE domains from NRPSs have demonstrated the power of this technique to generate libraries of structurally diverse cyclic non-ribosomal peptides, as well as cyclic polyketide-non-ribosomal pepCurrent Opinion in Biotechnology 2006, 17:597–605
tides [40,41]. Similar efforts are also being pursued with PKS systems. The Pik TE is known to possess a relaxed specificity, as it is capable of accepting and cyclizing both hexaketide and heptaketide intermediates [42]. To investigate the use of the Pik TE as a macrolactonization catalyst, Aldrich et al. [43] have recently described an efficient synthesis of the hexaketide intermediate in the pikromycin biosynthetic pathway, and the subsequent in vitro cyclization of this intermediate using the excised TE domain to yield 10-deoxymethynolide. This initial effort offers the hope that the Pik TE could be used to generate a library of structurally diverse 12- and 14-membered ring macrolactones from synthetic substrate analogs. However, this promise is based on the assumption that the enzyme will be able to accommodate a variety of substrates. At present, work is focused on achieving an enhanced understanding of Pik TE specificity. To this end, Fecik and co-workers [44] have demonstrated that diphenylphosphonate triketides can effectively serve as Pik TE affinity labels, offering a valuable tool for the examination of the substrate specificity and catalytic mechanism of this PKS domain. Indeed, recent structural studies of Pik TE bound with an extended chain diphenylphosphonate www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 603
Figure 4
Structure of the pikromycin TE domain modified with a heptaketide mimic affinity label. The active site serine residue 148 of the pikromycin TE is covalently attached to the affinity label. It is proposed that the two ordered water molecules that are coordinated between Gln 183 and Ala 217 form a ‘hydrophilic barrier’, serving to redirect the linear substrate back into the substrate channel. This, coupled with a hydrogen bond interaction with Thr 77, steers the affinity label into the observed curled conformation. PDB coordinates 2HFK [45] were used to generate this figure.
heptaketide mimic have yielded remarkable insight into both of these issues (Figure 4) [45]. Specifically, the paucity of direct enzyme–substrate interactions implies that Pik TE could tolerate structurally diverse substrate analogs; however, it is likely that the preservation of the a/b unsaturated ketone functionality is required for efficient cyclization [45]. Based on the success of these studies, affinity labels are poised to become increasingly effective tools for elucidating mechanisms and substrate specificities of other PKS domains, thereby providing novel insights that will enable the construction of functional and efficient hybrid PKS systems.
Although advances over the past few years have significantly increased our ability to successfully construct hybrid PKS pathways, important challenges remain before the power of this approach is realized. Current and future efforts that focus on enhancing our understanding of substrate specificity, kinetics and rate-limiting steps in modular polyketide biosynthesis will undoubtedly benefit efforts to rationally recombine PKS catalytic units into hybrid systems. Furthermore, an increase in protein structural knowledge of modular PKS architecture, utilizing both X-ray crystallography and NMR approaches, is absolutely crucial to these efforts.
Conclusions
Update
The manipulation and recombination of modular PKS systems offers an exciting strategy for the generation of structurally diverse libraries of novel natural products that have potential medicinal value. However, because of the lack of detailed molecular understanding of domain and module interactions within PKS systems, the promise of this technology has remained largely unrealized.
Together, the Noel and Kay laboratories have recently described a novel, naturally occurring hybrid PKS biosynthetic system from Dictystelium discoideum in which a type I FAS is covalently tethered to an iterative type III PKS (Steely2) [46]. Within this unique protein architecture, the type III PKS serves as a replacement for the native C-terminal TE domain of the FAS. Thus, it is
www.sciencedirect.com
Current Opinion in Biotechnology 2006, 17:597–605
604 Chemical biotechnology
presumed that, much like a type I PKS module, the Steely2 type III PKS accepts substrates by direct transfer from the upstream acyl-ACP, or indirectly as an acyl-CoA following off-loading from the FAS terminal ACP domain. This unprecedented association of two independent catalytic systems offers an unexplored strategy for the synthesis of chemically diverse polyketide compounds. Given the enhanced appreciation for the structural relatedness between type I PKSs and FASs [47], the two natural FAS–PKS hybrids from D. discoideum provide a working model for the functional recombination of type I PKS modules with type III PKS systems, furthering the potential of hybrid polyketide synthetic pathways.
13. Reeves CD, Murli S, Ashley GW, Piagentini M, Hutchinson CR, McDaniel R: Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry 2001, 40:15464-15470.
Acknowledgements
17. Patel K, Piagentini M, Rascher A, Tian ZQ, Buchanan GO, Regentin R, Hu ZH, Hutchinson CR, McDaniel R: Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition. Chem Biol 2004, 11:1625-1633.
The authors gratefully acknowledge David Akey for assistance in generating Figures 3 and 4 and Tonia Buchholz for critical review of the manuscript. The Sherman laboratory is generously supported by grants from the National Institutes of Health (GM076477, CA108874, TW007404), the Hans and Ella McCollum Vahlteich Research Fund at the University of Michigan College of Pharmacy and the J.G. Searle Professorship in Medicinal Chemistry. JDK is supported by an NRSA postdoctoral fellowship (F32 GM075641) from the NIH.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Newman DJ, Cragg GM, Snader KM: The influence of natural products upon drug discovery. Nat Prod Rep 2000, 17:215-234.
2.
Newman DJ, Cragg GM, Snader KM: Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 2003, 66:1022-1037.
3.
O’Hagan D: Evolution of the polyketide metabolites. Chichester, UK: Ellis Horwood; 1995.
4.
Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L: Modular organization of genes required for complex polyketide biosynthesis. Science 1991, 252:675-679.
5.
Hill AM: The biosynthesis, molecular genetics and enzymology of the polyketide-derived metabolites. Nat Prod Rep 2006, 23:256-320.
6.
Staunton J, Weissman KJ: Polyketide biosynthesis: a millennium review. Nat Prod Rep 2001, 18:380-416.
7.
Walsh CT: Combinatorial biosynthesis of antibiotics: challenges and opportunities. ChemBioChem 2002, 3:125-134.
8.
Walsh CT: Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 2004, 303:1805-1810.
9.
Fortman JL, Sherman DH: Utilizing the power of microbial genetics to bridge the gap between the promise and the application of marine natural products. ChemBioChem 2005, 6:960-978.
10. McDaniel R, Welch M, Hutchinson CR: Genetic approaches to polyketide antibiotics. 1. Chem Rev 2005, 105:543-558.
14. Del Vecchio F, Petkovic H, Kendrew SG, Low L, Wilkinson B, Lill R, Cortes J, Rudd BAM, Staunton J, Leadlay PF: Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol 2003, 30:489-494. 15. Kumar P, Koppisch AT, Cane DE, Khosla C: Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA: ACP transacylase. J Am Chem Soc 2003, 125:14307-14312. 16. Hans M, Hornung A, Dziarnowski A, Cane DE, Khosla C: Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules. J Am Chem Soc 2003, 125:5366-5374.
18. Kim CY, Alekseyev VY, Chen AY, Tang YY, Cane DE, Khosla C: Reconstituting modular activity from separated domains of 6deoxyerythronolide B synthase. Biochemistry 2004, 43:1389213898. A stable KS–AT di-domain, identified by limited proteolysis, is reported to be capable of catalyzing chain elongation when presented with an excised ACP domain, suggesting the importance of maintaining the native KS–AT domain interface in hybrid PKS engineering. 19. Chen AY, Schnarr NA, Kim CY, Cane DE, Khosla C: Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. J Am Chem Soc 2006, 128:3067-3074. 20. Tang Y, Kim CY, Mathews II, Cane DE, Khosla C: The 2.7 A˚ crystal structure of a 194-kDa homodimeric fragment of the 6deoxyerythronolide B synthase. Proc Natl Acad Sci USA 2006, 103:11124-11129. An enhanced understanding of domain organization within type I PKS modules is provided by the crystal structure of the KS–AT di-domain polypeptide fragment from DEBS module 5. This structure is consistent with the recently elucidated FAS structure, further emphasizing the relatedness in the biosynthesis between type I polyketides and fatty acids. 21. Zhang X, Chen Z, Li M, Wen Y, Song Y, Li J: Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol 2006, in press (http://dx.doi.org/10.1007/s00253-006-0361-2). 22. Maier T, Jenni S, Ban N: Architecture of mammalian fatty acid synthase at 4.5 angstrom resolution. Science 2006, 311:1258-1262. The X-ray structure of the mammalian FAS is reported, the first elucidated for any megasynthase. Given the similarities between PKSs and FASs, this structure offers an organizational template for domain interactions within PKS systems. 23. Jenni S, Leibundgut M, Maier T, Ban N: Architecture of a fungal fatty acid synthase at 5 A˚ resolution. Science 2006, 311:1263-1267. 24. Tsai SC, Miercke LJW, Krucinski J, Gokhale R, Chen JCH, Foster PG, Cane DE, Khosla C, Stroud RM: Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc Natl Acad Sci USA 2001, 98:14808-14813.
11. Weissman KJ, Leadlay PF: Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol 2005, 3:925-936. A detailed review highlighting successes and challenges in combinatorial biosynthetic strategies involving modular polyketide synthases.
25. Tsai SC, Lu HX, Cane DE, Khosla C, Stroud RM: Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. Biochemistry 2002, 41:12598-12606.
12. Gonzalez-Lergier J, Broadbelt LJ, Hatzimanikatis V: Theoretical considerations and computational analysis of the complexity in polyketide synthesis pathways. J Am Chem Soc 2005, 127:9930-9938.
26. Keatinge-Clay AT, Stroud RM: The structure of a ketoreductase determines the organization of the b-carbon processing enzymes of modular polyketide synthases. Structure 2006, 14:737-748.
Current Opinion in Biotechnology 2006, 17:597–605
www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 605
This article describes the first X-ray structure of a PKS reductive domain, the ketoreductase from module 1 of the DEBS PKS. Knowledge of the KR domain boundaries will be beneficial to future PKS redesign experiments that attempt to install b-keto processing domains.
37. Tsuji SY, Cane DE, Khosla C: Selective protein–protein interactions direct channeling of intermediates between polyketide synthase modules. Biochemistry 2001, 40:2326-2331.
27. Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, Santi DV: Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci USA 2004, 101:15573-15578. A novel approach for the accurate and efficient synthesis of large DNA fragments is described that enables the reconstruction of polyketide synthase gene clusters with optimized codon usage and with rationally incorporated restriction sites for downstream engineering of PKS hybrids.
38. Weissman KJ: The structural basis for docking in modular polyketide biosynthesis. ChemBioChem 2006, 7:485-494. The author examines the protein–protein interactions that exist within PKS systems and suggests that binding specificity can be altered by simply exchanging the docking domain helices that are involved in the four-a-helix bundle. Furthermore, this work proposes that specific amino acid residues (code residues) contribute to the specificity of docking domain partners.
28. Kodumal SJ, Santi DV: DNA ligation by selection. Biotechniques 2004, 37:34-40.
39. Kim BS, Sherman DH, Reynolds KA: An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases. Protein Eng Des Sel 2004, 17:277-284.
29. Menzella HG, Reisinger SJ, Welch M, Kealey JT, Kennedy J, Reid R, Tran CQ, Santi DV: Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster. J Ind Microbiol Biotechnol 2006, 33:22-28. 30. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA, Santi DV: Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 2005, 23:1171-1176. This paper describes the codon-optimized synthesis of 14-chain elongation modules from eight different PKS systems and reports their assembly into 154 bi-modular combinations, of which over half generated the predicted product. This work represents a tremendous advance in the development of hybrid PKS systems. 31. Sherman DH: The Lego-ization of polyketide biosynthesis. Nat Biotechnol 2005, 23:1083-1084. 32. Chandran SS, Menzella HG, Carney JR, Santi DV: Activating hybrid modular interfaces in synthetic polyketide synthases by cassette replacement of ketosynthase domains. Chem Biol 2006, 13:469-474. The restoration of activity to chemically incompetent bi-modular PKS hybrids through replacement and installation of the ketosynthase domain that natively interacts with the upstream ACP domain is reported. This work emphasizes the importance of ACP–KS interactions in the transfer of the growing polyketide chain. 33. Gokhale RS, Tsuji SY, Cane DE, Khosla C: Dissecting and exploiting intermodular communication in polyketide synthases. Science 1999, 284:482-485. 34. Hahn M, Stachelhaus T: Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proc Natl Acad Sci USA 2004, 101:15585-15590. 35. Gokhale RS, Khosla C: Role of linkers in communication between protein modules. Curr Opin Chem Biol 2000, 4:22-27. 36. Broadhurst RW, Nietlispach D, Wheatcroft MP, Leadlay PF, Weissman KJ: The structure of docking domains in modular polyketide synthases. Chem Biol 2003, 10:723-731.
www.sciencedirect.com
40. Kohli RM, Walsh CT, Burkart MD: Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature 2002, 418:658-661. 41. Kohli RM, Burke MD, Tao JH, Walsh CT: Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules. J Am Chem Soc 2003, 125:7160-7161. 42. Xue Y, Sherman DH: Alternative modular polyketide synthase expression controls macrolactone structure. Nature 2000, 403:571-575. 43. Aldrich CC, Venkatraman L, Sherman DH, Fecik RA: Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide. J Am Chem Soc 2005, 127:8910-8911. 44. Giraldes JW, Akey DL, Kittendorf JD, Sherman DH, Smith JL, Fecik RA: Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels. Nat Chem Biol 2006, 2:531-536. 45. Akey DL, Kittendorf JD, Giraldes JW, Fecik RA, Sherman DH, Smith JL: Structural basis for macrocyclization by the pikromycin thioesterase. Nat Chem Biol 2006, 2:537-542. As a follow up to [44], this paper describes the application of enzyme affinity labels toward the investigation of the substrate specificity and catalytic mechanism of the Pik PKS TE domain, and also reports the cocrystal structure of the Pik TE bound with its product, 10-deoxymethynolide. The results from these studies reveal a novel mechanism for macrolactone formation. 46. Austin MB, Saito T, Bowman ME, Haydock S, Kato A, Moore BS, Kay RR, Noel JP: Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase. Nat Chem Biol 2006, 2:494-502. 47. Sherman DH, Smith JL: Clearing the skies over modular polyketide synthases. ACS Chem Biol 2006, 1:505-509.
Current Opinion in Biotechnology 2006, 17:597–605
Current Opinion in Biotechnology 2006, 17:597–605 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 12th October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.09.005
Introduction Historically, nature has been a rich source of structurally complex organic molecules that have proven beneficial to human health [1]. Currently, there are many examples of natural product-derived pharmaceuticals that are employed to combat human disease [2], and those classified as polyketides are among the most important. Polyketides represent a large class of microbial and plant-derived metabolites that display a dazzling combination of structural diversity and pharmacological activity [3]. These organic molecules vary in molecular weight, functional group modification and complexity, and include linear, polycyclic and macrocyclic structural forms. Most remarkable, their medicinal impact spans a growing range of human and animal diseases. At present, polyketide-based pharmaceuticals find clinical utility as www.sciencedirect.com
antibiotics, antiparasitic agents, antifungals, anticancer drugs and immunosuppressants (Figure 1). Because of their therapeutic potential, an ever-increasing demand has been placed on current natural product research to uncover novel polyketide compounds. Although their discovery continues to proceed at a rapid pace, the clinical development of these compounds remains constrained by the fact that their isolation from natural hosts is often limited in scale. Furthermore, the structural complexity displayed by this class of compounds poses substantial challenges for the total synthesis of the target natural product and its analogs. These limitations have significantly hindered the efforts to modulate the desired combination of pharmacological, pharmacokinetic and pharmacodynamic properties of lead compounds. Recent efforts to increase both the yield and the structural diversity of polyketide natural products have focused on harnessing the catalytic power and specificity of the enzymes responsible for polyketide biosynthesis. The biosynthesis of macrolide polyketides relies on type I modular polyketide synthases (PKSs) to catalyze the stepwise condensation of simple carboxylic acid derivatives (e.g. malonyl-CoA and methylmalonyl-CoA). Organizationally, type I PKSs are arranged into modules, wherein each module is comprised of a set of catalytic activities that is responsible for a single elongation of the polyketide chain, and subsequent reductive processing of the b-keto functionality (Figure 2) [4]. The sequential arrangement of modules within a PKS system effectively serves as a biosynthetic assembly line, responsible for dictating the final size and structure of the polyketide scaffold. Readers interested in additional background information regarding PKS systems are referred to one of the many recent reviews that have been written on this topic [5–9]. The modular nature of type I PKSs offers several strategies for the generation of structurally diverse polyketides through a combinatorial ‘plug and play’ approach [10,11]. For example, the alteration of individual catalytic domains, via inactivation, substitution, addition or deletion, can yield predicted structural alterations of the final PKS product. Likewise, the addition, deletion or exchange of intact modules can also impart structural variety into polyketide metabolites. Using these and other approaches, over 200 novel structures have been generated. Although impressive, these successes appear to be more the exception rather than the rule, as many Current Opinion in Biotechnology 2006, 17:597–605
598 Chemical biotechnology
Figure 1
Examples of clinically important polyketides.
efforts result in trace levels, or fail to produce the desired product. A recent theoretical analysis suggests that modular PKSs have the potential to generate hundreds of millions of compounds through the novel assembly of catalytic units [12]; however, it has become increasingly apparent that before this potential can be met, much remains to be learned regarding the intricacies of domain and modular interactions within PKS systems. In this review, recent advances in the understanding of these issues are highlighted, and their application toward the construction of hybrid PKS systems is discussed.
Manipulation of PKS catalytic domains It is well established that the arrangement of catalytic domains within a PKS module directly influences polyketide structure [4]. As such, the manipulation of catalytic domains presents an attractive strategy for the generation of novel chemotypes. At a minimum, each PKS elongation module contains an acyl transferase (AT) Current Opinion in Biotechnology 2006, 17:597–605
domain, an acyl carrier protein (ACP) domain and a ketosynthase (KS) domain (Figure 2). The AT is responsible for specifying and loading the appropriate CoA extender unit onto the ACP (e.g. malonyl-CoA, methylmalonyl-CoA or methoxymalonyl-CoA). The KS domain then catalyzes a decarboxylative condensation of the extender unit with the growing polyketide chain obtained from the preceding module to generate an ACP-bound b-ketoacyl product. In addition to these three core domains, each elongation module can contain up to three additional domains that are responsible for reductive processing of the b-keto functionality before the next extension step. The presence of a ketoreductase (KR) domain gives rise to a b-hydroxyl functionality; the presence of both a KR and a dehydratase (DH) domain generates an alkene, whereas the combination of KR, DH and enoyl reductase (ER) results in complete reduction to an alkane. Finally, termination of polyketide biosynthesis is catalyzed by a thioesterase (TE) domain www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 599
Figure 2
The pikromycin PKS: an example of a modular polyketide synthase. The pikromycin PKS contains one loading module and six elongation modules spanning four polypeptide chains (PikAI–PikAIV). Chain elongation through PikAIII followed by TE-catalyzed termination results in the 12-membered macrolactone product, 10-deoxymethynolide, whereas continued elongation of the polyketide chain produces the 14-membered macrolactone, narbonolide. Both products undergo further processing via a glycosyl transferase and P450 hydroxylase to yield the methymycin and pikromycin suite of antibiotics. Abbreviations: ACP, acyl carrier protein; AT, acyl transferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KR0, inactive ketoreductase; KS, ketosynthase; KSQ, decarboxylase; TE, thioesterase.
located at the carboxy terminus of the final elongation module (Figure 2). Alteration of PKS catalytic domains can provide access to structural diversity that is not easily achieved via standard synthetic methodology. For example, genetic manipulation of AT domains can effectively modify the carbon substituents that decorate the polyketide scaffold. Typically, the specificity of an AT domain (e.g. methylmalonyl-CoA versus malonyl-CoA) can be altered via sitedirected mutagenesis [13,14]. Alternatively, inactivated ATs can be complemented via exogenous AT-like enzymes acting in trans. Khosla and co-workers [15] have demonstrated that the completely inactivated AT domain www.sciencedirect.com
from module 6 of the 6-deoxyerythronolide B (DEBS) PKS can be rescued with a heterologous malonylCoA:ACP transacylase (MAT) to efficiently generate 2-desmethyl-6-deoxyerythronolide B. AT domains can also be genetically swapped between PKS systems [16]; however, this often results in reduced catalytic efficiency of the hybrid multifunctional protein, a phenomenon that has been attributed to an overall structural perturbation of the module. Most recently, researchers at Kosan Biosciences [17] have reportedly replaced the AT domains in six of the seven geldanamycin PKS modules with a malonyl-specific AT from the rapamycin biosynthetic cluster. Remarkably, four of the six hybrid modules successfully produced the predicted geldanamycin Current Opinion in Biotechnology 2006, 17:597–605
600 Chemical biotechnology
analogs, albeit in much lower yield relative to geldanamycin in the producing strain. The use of inaccurate domain boundaries presumably destabilizes the modular architecture, resulting in the two nonfunctional hybrids.
it is expected that this newfound structural information can be leveraged into more successful strategies for the incorporation of functional reductive domains into hybrid PKS systems.
It has recently been reported that limited proteolysis of DEBS module 3 produces a stable peptide fragment consisting of a KS–AT di-domain [18]. Subsequently, KS–AT di-domains from DEBS modules 3 and 6 have been cloned and overexpressed [18,19]. When presented with a separate ACP domain, the recombinant KS–AT di-domains demonstrate the ability to catalyze condensation activity, suggesting that maintenance of the KS–AT interface is crucial in the design of hybrid PKS modules. An enhanced understanding of this interaction has recently been gained from the crystal structure of the recombinant KS–AT di-domain from DEBS module 5 [20]. On the basis of this structure, it appears that the relative positions of the KS and AT domains are rigidly fixed via interactions with both the interdomain linker region and the linker region immediately downstream of the AT. This suggests that in domain exchange experiments, the accurate dissection and insertion of the AT is likely crucial for the maintenance of a strict spatial organization between KS and AT. Thus, structural disruption might be best minimized in a hybrid module via simultaneous swapping of native KS–AT di-domain pairs to preserve the natural interface; however, this approach remains to be experimentally explored.
DNA redesign: increasing the modularity of the PKS module
The incorporation of reductive processing domains into PKS modules can also lead to the generation of novel chemotypes [21]. As with the exchange of AT domains, the successful addition of reductive domains into PKS modules often results in attenuated catalytic efficiency. Currently, the lack of available structural information of PKSs precludes definitive identification of domain boundaries, thus presenting a major barrier for successful construction of hybrid PKS systems. Crystal structures of two fatty acid synthases (FASs, mammalian and fungal) have recently been reported [22,23]. Although the similarity that exists between FASs and PKSs offers hope that a structure for a PKS module is forthcoming, current efforts are focused on determining the structures of individual PKS domains. In addition to the previously described structure of the DEBS module 5 KS–AT di-domain [20], the structures of the TE domain from the DEBS PKS and pikromycin (Pik) PKS have been solved [24,25]. Most recently, Stroud and co-workers [26] have reported the structure of the KR domain (in the presence of an NADPH cofactor) from module 1 of the DEBS PKS, providing important insights into key structural details. From these data, the domain boundaries of the KR domain are readily defined. Moreover, an enhanced understanding of the domain interfaces that exist between all three reductive domains relative to ACP is provided. As such, Current Opinion in Biotechnology 2006, 17:597–605
The high percentage of G+C content, which is often characteristic of actinomycetes-derived PKS-encoding genes, makes genetic manipulation via standard molecular biology a challenging endeavor. To overcome this issue, a strategy has been developed for the efficient synthesis of long, contiguous fragments of DNA [27]. The appeal of this semi-synthetic technology is twofold. First, it allows for the reprogramming of the codon usage of a given PKS gene to one that is more amenable to gene expression in heterologous hosts, such as Escherichia coli. Second, it enables the incorporation of rationally positioned restriction enzyme recognition sequences, making downstream genetic exchange a much less complicated process. This technology relies on the chemical synthesis of 500 bp DNA ‘synthons’ that are subsequently assembled into 5 kb ‘multisynthons’ via the ligation by selection (LBS) method [28]. As proof of concept, the complete DEBS-encoding gene cluster has been redesigned, synthesized and expressed in E. coli [29]. The streamlined redesign and synthesis of large DNA fragments offers a general approach for the construction of hybrid PKS modules. Menzella et al. [30] have recently described a powerful strategy in which synthetic building blocks (i.e. catalytic domains or modules) from a variety of PKS systems can be synthetically engineered to incorporate unique restriction sites flanking either end. These engineered sites enable the facile plug and play recombination of catalytic domains and modules, a technique that has been referred to as the ‘Lego-ization’ of polyketide biosynthesis [31]. The general applicability of this approach was demonstrated by synthesizing 14 different modules from eight different PKS systems and rearranging them into 154 different bi-modular combinations, suitable for E. coli expression. Notably, just under half of the bi-modules generated the predicted triketide lactone product. However, each of the recombined modules demonstrated catalytic competence by processing unnatural substrates. This suggests that the lack of activity displayed by some bi-module combinations is most likely due to faulty communication between the upstream ACP and the downstream KS of interacting modules. Indeed, Santi and co-workers [32] have recently described the activation of nonproductive bimodular hybrids through KS domain exchange, whereby the KS domain of the acceptor module was replaced with the KS domain that naturally interacts with the upstream ACP. This success underscores the importance of optimizing modular interactions in the future design of hybrid PKS pathways. www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 601
Module exchange Perhaps the most obvious strategy for the construction of hybrid polyketide synthetic pathways is via de novo assembly of individual modules from a variety of PKS systems. The appeal of this approach lies in the fact that the enzymatic domains within a given PKS module have already been optimized to work as a contiguous unit. As such, structural perturbations that translate into catalytic inefficiency should be minimized. Chemical diversity can be implemented into the polyketide scaffold by assembling modules that contain the desired extender unit specificity and a combination of reductive domains without concern for optimizing domain interactions within any given module. However, the feasibility of this approach requires (i) efficient and specific communication between interacting ‘heterologous’ modules to facilitate channeling of the polyketide intermediates and (ii) broad substrate tolerance of the individual domains that comprise the heterologous system. Failure to meet these requirements would inevitably result in a significant barrier for the production of novel polyketides via hybrid PKS systems. The assembly-line mechanism employed by modular PKSs relies on the efficient transfer of linear intermediates between consecutive elongation modules. Specifically, the ACP-bound intermediate of the upstream module must be channeled to the downstream KS domain before initiating another round of chain elongation. For modules contained on a single PKS polypeptide (e.g. bi-, tri- and tetra-modular PKSs), this interaction appears to be facilitated by short peptide linkers between consecutive ACP and KS domains [33]. By contrast, interactions between modules contained on separate polypeptides are mediated via longer peptide domains, termed ‘docking domains’, that are present at the C-terminus and N-terminus of the interacting polypeptides [33]. A similar type of communication occurs between interacting modules of non-ribosomal peptide synthases (NRPSs), offering the possibility to engineer novel PKS–NRPS hybrid pathways [34]. The inherent specificity that exists between PKS docking domain partners has previously been established [35]. Recent work has begun to elucidate the structural basis for this molecular recognition. Utilizing protein NMR spectroscopy, the structure of the docking domain interaction that exists between DEBS module 4 and DEBS module 5 was determined [36]. This structure demonstrates that the C-terminal docking domain of DEBS module 4 is comprised of three separate helical regions, whereas the N-terminal docking domain of DEBS module 5 consists of a single helix that adopts a coiled-coiled structure within the dimeric PKS. However, it appears that the interaction between these two docking domains is mediated solely by the terminal helix of the C-terminal docking domain (two per dimer) of the ACP and the www.sciencedirect.com
coiled-coiled dimeric structure of the N-terminal docking domain of the KS, essentially forming an intermolecular four-a-helix bundle (Figure 3). A survey of sequences from a variety of interacting PKS modules suggests that this structural motif is a universal feature of PKS docking domains [36]. The current model of docking domain interactions between PKS modules suggests that, as an alternative to the complementary exchanging of entire docking domains [37], communication between heterologous PKS modules could be achieved by simply installing a matched pair of the interacting helices that participate in the four-a-helix bundle. To further examine this, a series of experiments has been described in which the putative docking helices from interacting docking domains were exchanged, in either matched, mismatched or covalently linked pairs [38]. The substitution of these helices with a matched pair from another set of interacting modules did not yield a significant difference in product formation, whereas substitution with matched, covalently linked pairs gave a slight increase in product yields. As expected, the substitution with mismatched pairs resulted in decreased product yield. It has been proposed that specific interactions between individual amino acids (referred to as code residues) within the docking helices might serve to stabilize matched partners [38]. For example, the interaction between the putative code residues of the matched docking pairs of DEBS 2 and DEBS 3 is attractive (Arg and Asp), whereas in the mismatched pairs of DEBS 1 and DEBS 3, the interaction between these code residues is repulsive (Glu and Asp), likely contributing to their destabilization. Further experimentation is required to fully explore this idea; however, if the role of the code residues is found to be general, it might enable facile changes to interactions between PKS modules by simply changing a few amino acid residues.
Generation of hybrid PKSs via gene shuffling Gene shuffling techniques are routinely used in directed enzyme evolution experiments to impart specific properties and activities into an enzyme of interest; however, until recently, these techniques have never been applied to the construction of hybrid PKS systems. Using the Pik PKS system (Figure 2), Reynolds and coworkers [39] have described a method that utilizes in vivo recombination with a counter selectable marker, followed by functional complementation in Streptomyces venezuelae to generate a library of hybrid PikAI modules. A bioassay screen of over 4000 transformants identified three functional hybrids, underscoring the innate challenges in successful construction of hybrid PKS modules. Nonetheless, the application of this method to a variety of PKS modules lends itself as a powerful tool for the functional recombination of PKS domains and modules. Current Opinion in Biotechnology 2006, 17:597–605
602 Chemical biotechnology
Figure 3
Model of PKS docking domains. A model highlighting the four-a-helix bundle involved in mediating the protein–protein interaction between DEBS module 4 and DEBS module 5. Shown in blue are the terminal helices from C-terminal docking domain of module 4 and in green is the N-terminal docking domain of module 5. The putative ‘code residues’ (arginine and aspartate) that have been postulated to provide electrostatic stabilization to the docking interaction are highlighted. This figure was generated from the PDB coordinates 1PZR [36].
Polyketide diversity via chemoenzymatic synthesis: an alternative to hybrid PKS systems In theory, the construction of hybrid PKS systems offers a tremendous opportunity to access difficult-to-synthesize chemical entities via rational genetic manipulation [12]. Despite recent successes, the ability to achieve a productive outcome from any single experiment involving rational mixing and matching of components from a variety of PKS systems remains uncertain. As an alternative to hybrid PKSs, recent efforts have begun to focus on the development of chemoenzymatic strategies for the generation of new polyketide chemical diversity. Within this approach, structural diversity can be incorporated during the chemical synthesis of analogs based on advanced polyketide chain-elongation intermediates. These molecules are subsequently incorporated as substrates to either individual PKS modules (for elongation and processing) or individual PKS domains (e.g. thioesterase) to generate the final polyketide product. Several studies using excised TE domains from NRPSs have demonstrated the power of this technique to generate libraries of structurally diverse cyclic non-ribosomal peptides, as well as cyclic polyketide-non-ribosomal pepCurrent Opinion in Biotechnology 2006, 17:597–605
tides [40,41]. Similar efforts are also being pursued with PKS systems. The Pik TE is known to possess a relaxed specificity, as it is capable of accepting and cyclizing both hexaketide and heptaketide intermediates [42]. To investigate the use of the Pik TE as a macrolactonization catalyst, Aldrich et al. [43] have recently described an efficient synthesis of the hexaketide intermediate in the pikromycin biosynthetic pathway, and the subsequent in vitro cyclization of this intermediate using the excised TE domain to yield 10-deoxymethynolide. This initial effort offers the hope that the Pik TE could be used to generate a library of structurally diverse 12- and 14-membered ring macrolactones from synthetic substrate analogs. However, this promise is based on the assumption that the enzyme will be able to accommodate a variety of substrates. At present, work is focused on achieving an enhanced understanding of Pik TE specificity. To this end, Fecik and co-workers [44] have demonstrated that diphenylphosphonate triketides can effectively serve as Pik TE affinity labels, offering a valuable tool for the examination of the substrate specificity and catalytic mechanism of this PKS domain. Indeed, recent structural studies of Pik TE bound with an extended chain diphenylphosphonate www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 603
Figure 4
Structure of the pikromycin TE domain modified with a heptaketide mimic affinity label. The active site serine residue 148 of the pikromycin TE is covalently attached to the affinity label. It is proposed that the two ordered water molecules that are coordinated between Gln 183 and Ala 217 form a ‘hydrophilic barrier’, serving to redirect the linear substrate back into the substrate channel. This, coupled with a hydrogen bond interaction with Thr 77, steers the affinity label into the observed curled conformation. PDB coordinates 2HFK [45] were used to generate this figure.
heptaketide mimic have yielded remarkable insight into both of these issues (Figure 4) [45]. Specifically, the paucity of direct enzyme–substrate interactions implies that Pik TE could tolerate structurally diverse substrate analogs; however, it is likely that the preservation of the a/b unsaturated ketone functionality is required for efficient cyclization [45]. Based on the success of these studies, affinity labels are poised to become increasingly effective tools for elucidating mechanisms and substrate specificities of other PKS domains, thereby providing novel insights that will enable the construction of functional and efficient hybrid PKS systems.
Although advances over the past few years have significantly increased our ability to successfully construct hybrid PKS pathways, important challenges remain before the power of this approach is realized. Current and future efforts that focus on enhancing our understanding of substrate specificity, kinetics and rate-limiting steps in modular polyketide biosynthesis will undoubtedly benefit efforts to rationally recombine PKS catalytic units into hybrid systems. Furthermore, an increase in protein structural knowledge of modular PKS architecture, utilizing both X-ray crystallography and NMR approaches, is absolutely crucial to these efforts.
Conclusions
Update
The manipulation and recombination of modular PKS systems offers an exciting strategy for the generation of structurally diverse libraries of novel natural products that have potential medicinal value. However, because of the lack of detailed molecular understanding of domain and module interactions within PKS systems, the promise of this technology has remained largely unrealized.
Together, the Noel and Kay laboratories have recently described a novel, naturally occurring hybrid PKS biosynthetic system from Dictystelium discoideum in which a type I FAS is covalently tethered to an iterative type III PKS (Steely2) [46]. Within this unique protein architecture, the type III PKS serves as a replacement for the native C-terminal TE domain of the FAS. Thus, it is
www.sciencedirect.com
Current Opinion in Biotechnology 2006, 17:597–605
604 Chemical biotechnology
presumed that, much like a type I PKS module, the Steely2 type III PKS accepts substrates by direct transfer from the upstream acyl-ACP, or indirectly as an acyl-CoA following off-loading from the FAS terminal ACP domain. This unprecedented association of two independent catalytic systems offers an unexplored strategy for the synthesis of chemically diverse polyketide compounds. Given the enhanced appreciation for the structural relatedness between type I PKSs and FASs [47], the two natural FAS–PKS hybrids from D. discoideum provide a working model for the functional recombination of type I PKS modules with type III PKS systems, furthering the potential of hybrid polyketide synthetic pathways.
13. Reeves CD, Murli S, Ashley GW, Piagentini M, Hutchinson CR, McDaniel R: Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry 2001, 40:15464-15470.
Acknowledgements
17. Patel K, Piagentini M, Rascher A, Tian ZQ, Buchanan GO, Regentin R, Hu ZH, Hutchinson CR, McDaniel R: Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition. Chem Biol 2004, 11:1625-1633.
The authors gratefully acknowledge David Akey for assistance in generating Figures 3 and 4 and Tonia Buchholz for critical review of the manuscript. The Sherman laboratory is generously supported by grants from the National Institutes of Health (GM076477, CA108874, TW007404), the Hans and Ella McCollum Vahlteich Research Fund at the University of Michigan College of Pharmacy and the J.G. Searle Professorship in Medicinal Chemistry. JDK is supported by an NRSA postdoctoral fellowship (F32 GM075641) from the NIH.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Newman DJ, Cragg GM, Snader KM: The influence of natural products upon drug discovery. Nat Prod Rep 2000, 17:215-234.
2.
Newman DJ, Cragg GM, Snader KM: Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 2003, 66:1022-1037.
3.
O’Hagan D: Evolution of the polyketide metabolites. Chichester, UK: Ellis Horwood; 1995.
4.
Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L: Modular organization of genes required for complex polyketide biosynthesis. Science 1991, 252:675-679.
5.
Hill AM: The biosynthesis, molecular genetics and enzymology of the polyketide-derived metabolites. Nat Prod Rep 2006, 23:256-320.
6.
Staunton J, Weissman KJ: Polyketide biosynthesis: a millennium review. Nat Prod Rep 2001, 18:380-416.
7.
Walsh CT: Combinatorial biosynthesis of antibiotics: challenges and opportunities. ChemBioChem 2002, 3:125-134.
8.
Walsh CT: Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 2004, 303:1805-1810.
9.
Fortman JL, Sherman DH: Utilizing the power of microbial genetics to bridge the gap between the promise and the application of marine natural products. ChemBioChem 2005, 6:960-978.
10. McDaniel R, Welch M, Hutchinson CR: Genetic approaches to polyketide antibiotics. 1. Chem Rev 2005, 105:543-558.
14. Del Vecchio F, Petkovic H, Kendrew SG, Low L, Wilkinson B, Lill R, Cortes J, Rudd BAM, Staunton J, Leadlay PF: Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol 2003, 30:489-494. 15. Kumar P, Koppisch AT, Cane DE, Khosla C: Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA: ACP transacylase. J Am Chem Soc 2003, 125:14307-14312. 16. Hans M, Hornung A, Dziarnowski A, Cane DE, Khosla C: Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules. J Am Chem Soc 2003, 125:5366-5374.
18. Kim CY, Alekseyev VY, Chen AY, Tang YY, Cane DE, Khosla C: Reconstituting modular activity from separated domains of 6deoxyerythronolide B synthase. Biochemistry 2004, 43:1389213898. A stable KS–AT di-domain, identified by limited proteolysis, is reported to be capable of catalyzing chain elongation when presented with an excised ACP domain, suggesting the importance of maintaining the native KS–AT domain interface in hybrid PKS engineering. 19. Chen AY, Schnarr NA, Kim CY, Cane DE, Khosla C: Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. J Am Chem Soc 2006, 128:3067-3074. 20. Tang Y, Kim CY, Mathews II, Cane DE, Khosla C: The 2.7 A˚ crystal structure of a 194-kDa homodimeric fragment of the 6deoxyerythronolide B synthase. Proc Natl Acad Sci USA 2006, 103:11124-11129. An enhanced understanding of domain organization within type I PKS modules is provided by the crystal structure of the KS–AT di-domain polypeptide fragment from DEBS module 5. This structure is consistent with the recently elucidated FAS structure, further emphasizing the relatedness in the biosynthesis between type I polyketides and fatty acids. 21. Zhang X, Chen Z, Li M, Wen Y, Song Y, Li J: Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol 2006, in press (http://dx.doi.org/10.1007/s00253-006-0361-2). 22. Maier T, Jenni S, Ban N: Architecture of mammalian fatty acid synthase at 4.5 angstrom resolution. Science 2006, 311:1258-1262. The X-ray structure of the mammalian FAS is reported, the first elucidated for any megasynthase. Given the similarities between PKSs and FASs, this structure offers an organizational template for domain interactions within PKS systems. 23. Jenni S, Leibundgut M, Maier T, Ban N: Architecture of a fungal fatty acid synthase at 5 A˚ resolution. Science 2006, 311:1263-1267. 24. Tsai SC, Miercke LJW, Krucinski J, Gokhale R, Chen JCH, Foster PG, Cane DE, Khosla C, Stroud RM: Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc Natl Acad Sci USA 2001, 98:14808-14813.
11. Weissman KJ, Leadlay PF: Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol 2005, 3:925-936. A detailed review highlighting successes and challenges in combinatorial biosynthetic strategies involving modular polyketide synthases.
25. Tsai SC, Lu HX, Cane DE, Khosla C, Stroud RM: Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. Biochemistry 2002, 41:12598-12606.
12. Gonzalez-Lergier J, Broadbelt LJ, Hatzimanikatis V: Theoretical considerations and computational analysis of the complexity in polyketide synthesis pathways. J Am Chem Soc 2005, 127:9930-9938.
26. Keatinge-Clay AT, Stroud RM: The structure of a ketoreductase determines the organization of the b-carbon processing enzymes of modular polyketide synthases. Structure 2006, 14:737-748.
Current Opinion in Biotechnology 2006, 17:597–605
www.sciencedirect.com
Engineering polyketide synthetic pathways Kittendorf and Sherman 605
This article describes the first X-ray structure of a PKS reductive domain, the ketoreductase from module 1 of the DEBS PKS. Knowledge of the KR domain boundaries will be beneficial to future PKS redesign experiments that attempt to install b-keto processing domains.
37. Tsuji SY, Cane DE, Khosla C: Selective protein–protein interactions direct channeling of intermediates between polyketide synthase modules. Biochemistry 2001, 40:2326-2331.
27. Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, Santi DV: Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci USA 2004, 101:15573-15578. A novel approach for the accurate and efficient synthesis of large DNA fragments is described that enables the reconstruction of polyketide synthase gene clusters with optimized codon usage and with rationally incorporated restriction sites for downstream engineering of PKS hybrids.
38. Weissman KJ: The structural basis for docking in modular polyketide biosynthesis. ChemBioChem 2006, 7:485-494. The author examines the protein–protein interactions that exist within PKS systems and suggests that binding specificity can be altered by simply exchanging the docking domain helices that are involved in the four-a-helix bundle. Furthermore, this work proposes that specific amino acid residues (code residues) contribute to the specificity of docking domain partners.
28. Kodumal SJ, Santi DV: DNA ligation by selection. Biotechniques 2004, 37:34-40.
39. Kim BS, Sherman DH, Reynolds KA: An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases. Protein Eng Des Sel 2004, 17:277-284.
29. Menzella HG, Reisinger SJ, Welch M, Kealey JT, Kennedy J, Reid R, Tran CQ, Santi DV: Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster. J Ind Microbiol Biotechnol 2006, 33:22-28. 30. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA, Santi DV: Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 2005, 23:1171-1176. This paper describes the codon-optimized synthesis of 14-chain elongation modules from eight different PKS systems and reports their assembly into 154 bi-modular combinations, of which over half generated the predicted product. This work represents a tremendous advance in the development of hybrid PKS systems. 31. Sherman DH: The Lego-ization of polyketide biosynthesis. Nat Biotechnol 2005, 23:1083-1084. 32. Chandran SS, Menzella HG, Carney JR, Santi DV: Activating hybrid modular interfaces in synthetic polyketide synthases by cassette replacement of ketosynthase domains. Chem Biol 2006, 13:469-474. The restoration of activity to chemically incompetent bi-modular PKS hybrids through replacement and installation of the ketosynthase domain that natively interacts with the upstream ACP domain is reported. This work emphasizes the importance of ACP–KS interactions in the transfer of the growing polyketide chain. 33. Gokhale RS, Tsuji SY, Cane DE, Khosla C: Dissecting and exploiting intermodular communication in polyketide synthases. Science 1999, 284:482-485. 34. Hahn M, Stachelhaus T: Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proc Natl Acad Sci USA 2004, 101:15585-15590. 35. Gokhale RS, Khosla C: Role of linkers in communication between protein modules. Curr Opin Chem Biol 2000, 4:22-27. 36. Broadhurst RW, Nietlispach D, Wheatcroft MP, Leadlay PF, Weissman KJ: The structure of docking domains in modular polyketide synthases. Chem Biol 2003, 10:723-731.
www.sciencedirect.com
40. Kohli RM, Walsh CT, Burkart MD: Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature 2002, 418:658-661. 41. Kohli RM, Burke MD, Tao JH, Walsh CT: Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules. J Am Chem Soc 2003, 125:7160-7161. 42. Xue Y, Sherman DH: Alternative modular polyketide synthase expression controls macrolactone structure. Nature 2000, 403:571-575. 43. Aldrich CC, Venkatraman L, Sherman DH, Fecik RA: Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide. J Am Chem Soc 2005, 127:8910-8911. 44. Giraldes JW, Akey DL, Kittendorf JD, Sherman DH, Smith JL, Fecik RA: Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels. Nat Chem Biol 2006, 2:531-536. 45. Akey DL, Kittendorf JD, Giraldes JW, Fecik RA, Sherman DH, Smith JL: Structural basis for macrocyclization by the pikromycin thioesterase. Nat Chem Biol 2006, 2:537-542. As a follow up to [44], this paper describes the application of enzyme affinity labels toward the investigation of the substrate specificity and catalytic mechanism of the Pik PKS TE domain, and also reports the cocrystal structure of the Pik TE bound with its product, 10-deoxymethynolide. The results from these studies reveal a novel mechanism for macrolactone formation. 46. Austin MB, Saito T, Bowman ME, Haydock S, Kato A, Moore BS, Kay RR, Noel JP: Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase. Nat Chem Biol 2006, 2:494-502. 47. Sherman DH, Smith JL: Clearing the skies over modular polyketide synthases. ACS Chem Biol 2006, 1:505-509.
Current Opinion in Biotechnology 2006, 17:597–605