Genome Mining of Oxidation Modules in trans‐Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides

Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger‐type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans‐AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans‐AT PKSs in the specialized metabolism of symbiotic bacteria.     

Modular Oxime Formation by a trans-AT Polyketide Synthase

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.     

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H⁺-ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.     

Exploring the biosynthetic potential of bimodular aromatic polyketide synthases

Polycyclic aromatic polyketides such as actinorhodin and tetracenomycin are synthesized from acetate equivalents by type II polyketide synthases (PKS). Their carbon chain backbones are derived from malonyl-CoA building blocks through the action of a minimal PKS module consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and a malonyl-CoA/ACP transacylase. In contrast to these acetogenic polyketides, the backbones of a few aromatic polyketide natural products, such as the R1128 antibiotics, are primed by non-acetate building blocks. These polyketides are synthesized by bimodular PKSs comprising of a dedicated initiation module, which includes a ketosynthase, acyl transferase and ACP, as well as a minimal PKS module. Recently we showed that regioselectively modified polyketides could be synthesized through the genetic recombination of initiation modules and minimal PKS modules from different polyketide biosynthetic pathways (Tang et al. PLoS Biol. 2004, 2, 227-238). For example, the actinorhodin and tetracenomycin minimal PKSs could accept and elongate unnatural primer units from the R1128 initiation module. In this report we provide further examples of using heterologous bimodular PKSs for the engineered biosynthesis of new aromatic polyketides. In addition to providing insights into the biosynthetic mechanisms of aromatic PKSs, our findings also highlight considerable potential for crosstalk between amino acid catabolism and aromatic polyketide biosynthesis. For example, exogenously supplied unnatural amino acids are efficiently incorporated into bioactive anthraquinone antibiotics.     

Divergent Mechanistic Routes for the Formation of gem‐Dimethyl Groups in the Biosynthesis of Complex Polyketides

The gem‐dimethyl groups in polyketide‐derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem‐dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem‐dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem‐dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl‐ACP appeared to be the sole route to form a gem‐dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.     

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS⁰ shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.     

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum

Background: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete.Results: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea.Conclusions: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete–myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.     

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones

Enzymatic core components from trans‐acyltransferase polyketide synthases (trans‐AT PKSs) catalyze exceptionally diverse biosynthetic transformations to generate structurally complex bioactive compounds. Here we focus on a group of oxygenases identified in various trans‐AT PKS pathways, including those for pederin, oocydins, and toblerols. Using the oocydin pathway homologue (OocK) from Serratia plymuthica 4Rx13 and N‐acetylcysteamine (SNAC) thioesters as test surrogates for acyl carrier protein (ACP)‐tethered intermediates, we show that the enzyme inserts oxygen into β‐ketoacyl moieties to yield malonyl ester SNAC products. Based on these data and the identification of a non‐hydrolyzed oocydin congener with retained ester moiety, we propose a unified biosynthetic pathway of oocydins, haterumalides, and biselides. By providing access to internal ester, carboxylate pseudostarter, and terminal hydroxyl functions, oxygen insertion into polyketide backbones greatly expands the biosynthetic scope of PKSs.     

Rational design and assembly of synthetic trimodular polyketide synthases

Type I polyketide synthases (PKSs) consist of modules that add two-carbon units in polyketide backbones. Rearranging modules from different sources can yield novel enzymes that produce unnatural products, but the rules that govern module-module communication are still not well known. The construction and assay of hybrid bimodular units with synthetic PKS genes were recently reported. Here, we describe the rational design of trimodular PKSs by combining bimodular units. A cloning-expression system was developed to assemble and test 54 unnatural trimodular PKSs flanked by the loading module and the thioesterase from the erythromycin synthase. Remarkably, 96% of them produced the expected polyketide. The obtained results represent an important milestone toward the ultimate goal of making new bioactive polyketides by rational design. Additionally, these results show a path for the production of customized tetraketides by fermentation, which can be an important source of advanced intermediates to facilitate the synthesis of complex products.     

Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues.

BACKGROUND: Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules. CONCLUSIONS: Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.     

A Long‐Range Acting Dehydratase Domain as the Missing Link for C17‐Dehydration in Iso‐Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso‐migrastatin (iso‐MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module‐6, ‐9, ‐10 of MgsF (i.e., DH6, DH9, DH10) and module‐11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso‐MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β‐hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long‐range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.     

Molecular basis for acyl carrier protein–ketoreductase interaction in trans-acyltransferase polyketide synthases

The biosynthesis of polyketides by type I modular polyketide synthases (PKS) relies on co-ordinated interactions between acyl carrier protein (ACP) domains and catalytic domains within the megasynthase. Despite the importance of these interactions, and their implications for biosynthetic engineering efforts, they remain poorly understood. Here, we report the molecular details of the interaction interface between an ACP domain and a ketoreductase (KR) domain from a trans-acyltransferase (trans-AT) PKS. Using a high-throughput mass spectrometry (MS)-based assay in combination with scanning alanine mutagenesis, residues contributing to the KR-binding epitope of the ACP domain were identified. Application of carbene footprinting revealed the ACP-binding site on the KR domain surface, and molecular docking simulations driven by experimental data allowed production of an accurate model of the complex. Interactions between ACP and KR domains from trans-AT PKSs were found to be specific for their cognate partner, indicating highly optimised interaction interfaces driven by evolutionary processes. Using detailed knowledge of the ACP:KR interaction epitope, an ACP domain was engineered to interact with a non-cognate KR domain partner. The results provide novel, high resolution insights into the ACP:KR interface and offer valuable rules for future engineering efforts of biosynthetic assembly lines.

The interaction epitope between a cognate KR–ACP domain pairing from a trans-AT polyketide synthase is elucidated in molecular detail, providing unique insights into recognition and specificity of the interface.     

Toblerols: Cyclopropanol‐Containing Polyketide Modulators of Antibiosis in Methylobacteria

Trans‐AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans‐AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide‐ and cyclopropanol‐containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon–carbon shortening of a more extended precursor. Surprisingly, a knock‐out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild‐type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as‐yet unknown methylobacterial antibiotic.     

Assembly line termination in cylindrocyclophane biosynthesis: discovery of an editing type II thioesterase domain in a type I polyketide synthase

The termination step is an important source of structural diversity in polyketide biosynthesis. Most type I polyketide synthase (PKS) assembly lines are terminated by a thioesterase (TE) domain located at the C-terminus of the final module, while other PKS assembly lines lack a terminal TE domain and are instead terminated by a separate enzyme in trans. In cylindrocyclophane biosynthesis, the type I modular PKS assembly line is terminated by a freestanding type III PKS (CylI). Unexpectedly, the final module of the type I PKS (CylH) also possesses a C-terminal TE domain. Unlike typical type I PKSs, the CylH TE domain does not influence assembly line termination by CylI in vitro. Instead, this domain phylogenetically resembles a type II TE and possesses activity consistent with an editing function. This finding may shed light on the evolution of unusual PKS termination logic. In addition, the presence of related type II TE domains in many cryptic type I PKS and nonribosomal peptide synthetase (NRPS) assembly lines has implications for pathway annotation, product prediction, and engineering.     

Intermodular communication in modular polyketide synthases: structural and mutational analysis of linker mediated protein-protein recognition

Modular polyketide synthases (PKSs) present an attractive scaffold for the engineered biosynthesis of novel polyketide products via recombination of naturally occurring enzyme modules with desired catalytic properties. Recent studies have highlighted the pivotal role of short intermodular "linker pairs" in the selective channeling of biosynthetic intermediates between adjacent PKS modules. Using a combination of computer modeling, NMR spectroscopy, cross-linking, and site-directed mutagenesis, we have investigated the mechanism by which a linker pair from the 6-deoxyerythronolide B synthase promotes chain transfer. Our studies support a "coiled-coil" model in which the individual peptides comprising this linker pair adopt helical conformations that associate through a combination of hydrophobic and electrostatic interactions in an antiparallel fashion. Given the important contribution of such linker pair interactions to the kinetics of chain transfer between PKS modules, the ability to rationally modulate linker pair affinity by site-directed mutagenesis could be useful in the construction of optimized hybrid PKSs.     

Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase

BACKGROUND: Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest. RESULTS: The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains'). CONCLUSIONS: The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics.     

Structure-based dissociation of a type I polyketide synthase module

Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.     

Molecular recognition of diketide substrates by a β-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase

Background: Modular polyketide synthases (PKSs) are large multifunctional proteins that catalyze the biosynthesis of structurally complex bioactive products. The modular organization of PKSs has allowed the application of a combinatorial approach to the synthesis of novel polyketides via the manipulation of these biocatalysts at the genetic level. The inherent specificity of PKSs for their natural substrates, however, may place limits on the spectrum of molecular diversity that can be achieved in polyketide products. With the aim of further understanding PKS specificity, as a route to exploiting PKSs in combinatorial synthesis, we chose to examine the substrate specificity of a single intact domain within a bimodular PKS to investigate its capacity to utilize unnatural substrates.Results: We used a blocked mutant of a bimodular PKS in which formation of the triketide product could occur only via uptake and processing of a synthetic diketide intermediate. By introducing systematic changes in the native diketide structure, by means of the synthesis of unnatural diketide analogs, we have shown that the ketosynthase domain of module 2 (KS2 domain) in 6-deoxyerythronolide B synthase (DEBS) tolerates a broad range of variations in substrate structure, but it strongly discriminates against some others.Conclusions: Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.     

Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.