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.     

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.     

Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase

Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).     

Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.     

Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates

BACKGROUND: Polyketides are structurally diverse natural products with a range of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) 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 show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an unexpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construct a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketosynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprising. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisingly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively. CONCLUSIONS: These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.     

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.     

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.     

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.     

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.     

Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase

The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.     

Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase

BACKGROUND: Modular polyketide synthases catalyse the biosynthesis of medically useful natural products by stepwise chain assembly, with each module of enzyme activities catalysing a separate cycle of polyketide chain extension. Domain swapping between polyketide synthases leads to hybrid multienzymes that yield novel polyketides in a more or less predictable way. No experiments have so far been reported which attempt to enlarge a polyketide synthase by interpolating additional modules. RESULTS: We describe here the construction of tetraketide synthases in which an entire extension module from the rapamycin-producing polyketide synthase is covalently spliced between the first two extension modules of the erythromycin-producing polyketide synthase (DEBS). The extended polyketide synthases thus formed are found to catalyse the synthesis of specific tetraketide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzymes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaketide macrolactones. In each case the predicted products are accompanied by significant amounts of unextended products, corresponding to those of the unaltered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. CONCLUSIONS: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.     

Enzymatic Polyketide Chain Branching To Give Substituted Lactone,Lactam, and Glutarimide Heterocycles

Polyketides typically result from head‐to‐tail condensation of acyl thioesters to produce highly functionalized linear chains. The biosynthesis of the phytotoxin rhizoxin, however, involves a polyketide synthase (PKS) module that introduces a δ‐lactone chain branch through Michael addition of a malonyl extender to an α,β‐unsaturated intermediate unit. To evaluate the scope of the branching module, polyketide mimics were synthesized and their biotransformation by the reconstituted PKS module from the Rhizopus symbiont Burkholderia rhizoxinica was monitored in vitro. The impact of the type and configuration of the δ‐substituents was probed and it was found that amino‐substituted surrogates yield the corresponding lactams. A carboxamide analogue was transformed into a glutarimide unit, which can be found in many natural products. Our findings illuminate the biosynthesis of glutarimide‐bearing polyketides and also demonstrate the utility of this branching module for synthetic biology.     

Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules

Many polyketides are synthesized by a class of multifunctional enzymes called type I modular polyketide synthases (PKSs). Several reports have described the power of predictively altering polyketide structure by replacing individual PKS domains with homologues from other PKSs. For example, numerous erythromycin analogues have been generated by replacing individual methylmalonyl-specific acyl transferase (AT) domains of the 6-deoxyerythronolide B synthase (DEBS) with malonyl-, ethylmalonyl-, or methoxymalonyl-specific domains. However, the construction of hybrid PKS modules often attenuates product formation both kinetically and distributively. The molecular basis for this mechanistic imperfection is not understood. We have systematically analyzed the impact of replacing an AT domain of DEBS on acyl-AT formation, acyl-CoA:HS-NAc acyl transferase activity, acyl-CoA:ACP acyl transferase activity (nucleophile charging), acyl-SNAc:ketosynthase acyl transferase activity (electrophile charging), and beta-ketoacyl ACP synthase activity (condensation). As usual, domain junctions were located in interdomain regions flanking the AT domain. Kinetic analysis of hybrid modules containing either malonyl transferase or methylmalonyl transferase domains revealed a 15-20-fold decrease in overall turnover numbers of the hybrid modules as compared to the wild-type module. In contrast, both the activity and the specificity of the heterologous AT domains remained unaffected. Moreover, no defects could be detected in the ability of the heterologous AT domains to catalyze acyl-CoA:ACP acyl transfer. Single turnover studies aimed at directly probing the ketosynthase-catalyzed reaction led to two crucial findings. First, wild-type modules catalyzed chain elongation with comparable efficiency regardless of whether methylmalonyl-ACP or malonyl-ACP were the nucleophilic substrates. Second, chain elongation in all hybrid modules tested was seriously attenuated relative to the wild-type module. Our data suggest that, as currently practiced, the most deleterious impact of AT domain swapping is not on the substrate specificity. Rather, it is due to the impaired ability of the KS and ACP domains in the hybrid module to catalyze chain elongation. Consistent with this proposal, limited proteolysis of wild-type and hybrid modules showed major differences in cleavage patterns, especially in the region between the KR and ACP domains.     

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.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate ‘Streptomyces maritimus’: evidence for the derailment of an aromatic polyketide synthase

Background: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-β-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain ‘Streptomyces maritimus’ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from ‘S. maritimus’ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and (14)C-methylmalonyl-CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported values for the DEBS modules reveals significant differences between these systems.     

Enzymatic formation of unnatural novel polyketide scaffolds by plant-specific type III polyketide synthase

The catalytic potential of octaketide synthase (OKS), a plant-specific type III polyketide synthase (PKS) from Aloe arborescens, was investigated by phenylacetyl-CoA and benzoyl-CoA as starter substrates. As a result, a novel C₁₆ pentaketide coumarin was produced from phenylacetyl-CoA, whereas benzoyl-CoA was not a good substrate of OKS. Remarkably, a structure-guided OKS N222G mutant dramatically extended the product chain length to yield four novel polyketides including C₂₂ aromatic octaketides from the C₆-C₂ phenylacetyl starter, as well as a novel C₁₉ heptaketide benzophenone from the C₆-C₁ benzoyl starter.     

A Light-Activated Acyl Carrier Protein “Trap” for Intermediate Capture in Type II Iterative Polyketide Biocatalysis

A discrete acyl carrier protein (ACP) bearing a photolabile nonhydrolysable carba(dethia) malonyl pantetheine cofactor was chemoenzymatically prepared and utilised for the trapping of biosynthetic polyketide intermediates following light activation. From the in vitro assembly of the polyketides SEK4 and SEK4b, by the type II actinorhodin “minimal” polyketide synthase (PKS), a range of putative ACP-bound diketides, tetraketides, pentaketides and hexaketides were identified and characterised by FT-ICR-MS, providing direct insights on active site accessibility and substrate processing for this enzyme class.     

An Iterative Module in the Azalomycin F Polyketide Synthase Contains a Switchable Enoylreductase Domain

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension‐module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be “toggled” off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly‐line catalysis and may explain examples of apparent non‐colinearity in other modular PKS systems.     

Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 ? germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.