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.     

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.     

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.     

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.     

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.     

Iterative type II polyketide synthases,cyclases and ketoreductases exhibit context-dependent behavior in the biosynthesis of linear and angular decapolyketides

Background: Iterative type II polyketide synthases (PKSs) produce polyketide chains of variable but defined length from a specific starter unit and a number of extender units. They also specify the initial regiospecific folding and cyclization pattern of nascent polyketides either through the action of a cyclase (CYC) subunit or through the combined action of site-specific ketoreductase (KR) CYC CYC subunits. Additional CYCs and other modifications may be necessary to produce linear aromatic polyketides. The principles of the assembly of the linear aromatic polyketides, several of which are medically important, are well understood, but it is not clear whether the assembly of the angular aromatic (angucyclic) polyketides follows the same rules.Results: We performed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in comparison with their counterparts from the daunorubicin (dps) and tetracenomycin (tcm) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, the JadD and DpsF CYCs produced the same two polyketides, in the same ratio; neither product was angularly fused. The set of jadABCED PKS plus putative jadl CYC genes behaved similarly. Furthermore, no angular polyketides were isolated when the entire set of jad PKS enzymes and Jadl or the jad minimal PKS, Jadl and the TcmN CYC were present. The DpsE KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products.Conclusions: It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artificial gene cassettes under the control of a non-native promoter. The characteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving additional, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requirement and that CYC enzymes may act aberrantly as first-ring aromatases that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.     

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.     

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-beta-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.     

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.     

Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.     

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 gene cluster encoding resistomycin biosynthesis in Streptomyces resistomycificus; exploring polyketide cyclization beyond linear and angucyclic patterns

Resistomycin is a pentacyclic polyketide metabolite of Streptomyces resistomycificus that exhibits a variety of pharmacologically relevant properties. While virtually all bacterial aromatic polyketides can be grouped into linear and angular polyphenols, resistomycin has a unique "discoid" ring system. We have successfully identified the entire gene cluster encoding resistomycin biosynthesis by heterologously expressing a pooled cosmid library and screening for the fluorescence of the metabolite produced. The rem gene cluster exhibits several unusual features of the type II PKS involved, most remarkably a putative MCAT with highest homology to AT domains from modular PKSs. In addition, we provide the first insight into the molecular basis of a unique mode of cyclization giving rise to a discoid polyketide.     

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.     

Rational Design of Modular Polyketide Synthases: Morphing the Aureothin Pathway into a Luteoreticulin Assembly Line

The unusual nitro‐substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context‐dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non‐colinear behavior of PKS domains.     

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.     

Unprecedented mechanism of chain length determination in fungal aromatic polyketide synthases

Fungal aromatic polyketides show remarkable structural diversity fundamentally derived from variations in chain length and cyclization pattern. Their basic skeletons are synthesized by multifunctional iterative type I polyketide synthases (PKSs). Recently, we have found that the C-terminal thioesterase (TE)-like domain of Aspergillus nidulans WA catalyzes Claisen-type cyclization to form the B-ring of naphthopyrone YWA1. Here we report the unprecedented mechanism of chain length determination by the C-terminal TE-like domain of Colletotrichum lagenarium PKS1, which, in addition to catalyzing Claisen-type cyclization, intercepts the polyketomethylene intermediate from the acyl carrier protein domain during the condensation reaction to produce shorter chain length products. This chain length determination system is novel among PKSs, including bacterial and plant PKSs. The functional diversity of the TE-like domain directly influences the structural diversity of aromatic polyketides in C. lagenarium PKS1.     

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.     

Chemoenzymatic Synthesis of Spinosyn A

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post‐PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post‐PKS modifications.     

Context-dependent behavior of the enterocin iterative polyketide synthase; a new model for ketoreduction

Heterologous expression and mutagenesis of the enterocin type II polyketide synthase (PKS) system suggest for the first time that the association of an extended set of proteins and substrates is needed for the effective production of the enterocin-wailupemycin polyketides. In the absence of its endogenous ketoreductase (KR) EncD in either the enterocin producer "Streptomyces maritimus" or the engineered host S. lividans K4-114, the enterocin minimal PKS is unable to produce benzoate-primed polyketides, even when complemented with the homologous actinorhodin KR ActIII or with EncD active site mutants. These data suggest that the enterocin PKS requires EncD to serve a catalytic and not just a structural role in the functional PKS enzyme complex. This strongly implies that EncD reduces the polyketide chain during elongation rather than after its complete assembly, as suggested for most type II PKSs.     

Engineered biosynthesis of aklanonic acid analogues

Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.