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PubMed2005

Interaction between polyketide synthase and transporter suggests coupled synthesis and export of virulence lipid in M. tuberculosis.

Virulent mycobacteria utilize surface-exposed polyketides to interact with host cells, but the mechanism by which these hydrophobic molecules are transported across the cell envelope to the surface of the bacteria is poorly understood. Phthiocerol dimycocerosate (PDIM), a surface-exposed polyketide lipid necessary for Mycobacterium tuberculosis virulence, is the product of several polyketide synthases including PpsE. Transport of PDIM requires MmpL7, a member of the MmpL family of RND permeases. Here we show that a domain of MmpL7 biochemically interacts with PpsE, the first report of an interaction between a biosynthetic enzyme and its cognate transporter. Overexpression of the interaction domain of MmpL7 acts as a dominant negative to PDIM synthesis by poisoning the interaction between synthase and transporter. This suggests that MmpL7 acts in complex with the synthesis machinery to efficiently transport PDIM across the cell membrane. Coordination of synthesis and transport may not only be a feature of MmpL-mediated transport in M. tuberculosis, but may also represent a general mechanism of polyketide export in many different microorganisms.

Journal Article↗

PubMed2003

Creating polyketide diversity through genetic engineering.

Modular polyketide synthases (PKS) are large multifunctional enzymes that synthesize complex polyketides, a therapeutically important class of natural products. The linear order and composition of catalytic sites that comprise the PKS represent a "code" that determines the identity of the polyketide product. By re-programming the PKS through genetic engineering, it is possible to alter the code in a predictable manner to create specific structural modifications of polyketides and to produce new libraries of these natural products.

Carbohydrate Sequence↗

PubMed1994

Cloning and nucleotide sequence of a region of the Kibdelosporangium aridum genome homologous to polyketide biosynthetic genes.

The actinomycete Kibdelosporangium aridum naturally produces ardacin, a new glycopeptide antibiotic, the biosynthetic pathway of which should involve the participation of a polyketide synthase (PKS). A K. aridum 2.9 kb BamHI genomic fragment homologous to actI (a locus of the PKS cluster catalyzing polyketide chain assembly for actinorhodin biosynthesis in Streptomyces coelicolor) was isolated by shotgun cloning. This DNA fragment, called ardI, was sequenced and the deduced protein products were compared with those of other polyketide synthase genes, revealing similarities ranging from 50 to 80%. ardI was further used to probe a cosmid library of the K. aridum genome. Three hybridizing cosmids were obtained which contain overlapping inserts, together covering a 50 kb region, and including, 15 kb away from ardI, a fragment homologous to actIII, which codes for the ketoreductase of the actinorhodin PKS of S. coelicolor. All these findings indicate that at least part of a polyketide biosynthetic gene cluster has been isolated from the genome of the ardacin producer K. aridum.

Actinomycetales↗

PubMed1994

Characterization of polyketide ketoreductase gene (MPKR) from midecamycin-producing strain (Streptomyces mycarofaciens 1748).

This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis. A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S. galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant. The transformant produced aklavinone according to TLC and HPLC analyses. The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained. Introduction pCBR4 into S. galilaeus ATCC 31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter. Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S. galilaeus ATCC 31671. The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF). The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon. There was a 5nt (GGAGG) SD sequence at the upstream of start codon. A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region. The deduced amino acid sequences of MPKR gene consisted of 261 aa residues. Its amino acid were compared with actIII gene coding protein sequences. The similarity was 77.4% and the identity was 66.7%.

Amino Acid Sequence↗

PubMed2006

Amplification of DNA encoding entire type I polyketide synthase domains and linkers from streptomyces species.

Polyketides are a group of bioactive compounds from bacteria, plants, and fungi. To increase the availability of analogs for testing, the active sites of polyketide synthases are often substituted with homologous domains having altered substrate specificities. This study reports the design of polymerase chain reaction primers that enables isolation of entire active site domains from type I polyketide synthases with native interdomain linkers. This bypasses the need for further genetic screening to obtain functional units for use in genetic engineering. This is especially important in bioprospecting projects exploring new environments for bioresources.

Antifungal Agents↗

PubMed1994

Cloning, sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin.

A 10.2-kb fragment of DNA from Streptomyces roseofulvus, which contains polyketide synthase (PKS)-encoding genes (fren) presumed to determine production of the antibiotics frenolicin and the nanaomycins, was cloned. A 5530-bp continuous segment of this DNA was sequenced. Analysis of the sequence revealed five complete open reading frames (ORFs) transcribed in one direction (ORFs 1, 2, 3, 5, 4) and one (ORFX), located between ORF3 and ORF5, transcribed in the opposite direction. The deduced amino-acid sequences of ORFs 1, 2, 3, 4 and 5 closely resemble the sequences of known components of the type-II PKS from other Streptomyces species: putative heterodimeric (ORF1 + 2) ketosynthase, acyl carrier protein, cyclase and ketoreductase, respectively. A resemblance between the N-terminal and C-terminal halves of the ORF4 product--also discovered in the corresponding genes from other isochromanequinone antibiotic producers--suggests a possible origin of the cyclase-encoding gene by duplication. ORFX appears to represent a novel class of genes of unknown function present not only in the fren cluster, but also in other clusters of aromatic antibiotic biosynthetic genes in Streptomyces species. The fren-ORF1-5 genes, encoding a PKS that constructs a nascent polyketide of either 16 or 18 carbons, compared with fixed lengths of 16 and 20 for other available examples, are proving to be valuable for understanding the mechanisms controlling polyketide chain length and patterns of reduction and cyclisation.

Acyltransferases↗

PubMed2004

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.

Cyclization↗

PubMed2004

Novel mechanism for priming aromatic polyketide synthases.

In this issue of Chemistry & Biology, a novel priming mechanism is proposed for aromatic polyketide biosynthesis, with an iterative type I polyketide synthase generating a starter unit primed for a type II polyketide synthase. This novel priming system participates in hedamycin biosynthesis, a DNA alkylating agent.

Models, Molecular↗

PubMed2005

Biosynthesis of the antitumor agent chartreusin involves the oxidative rearrangement of an anthracyclic polyketide.

Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.

Antibiotics, Antineoplastic↗

PubMed2006

Activating hybrid modular interfaces in synthetic polyketide synthases by cassette replacement of ketosynthase domains.

Unnatural combinations of polyketide synthase modules often fail to make a polyketide product. The causes of these failures are likely complex and are not yet amenable to rational correction. One possible explanation is the inability of the ketosynthase (KS) domain to extend the ketide donated to it by the upstream module. We therefore addressed the problem by exchanging KS domains of the acceptor module in a combinatorial fashion and coexpressing these chimeric modules with ketide-donor modules that naturally interact with the transplanted KS. This approach was remarkably successful in activating previously unproductive bimodular combinations, and the results augur well for the ongoing development of molecular tools to design and produce novel polyketides.

Ligases↗

PubMed2006

Developing tools for engineering hybrid polyketide synthetic pathways.

Bacterial type I polyketide synthases (PKSs) are complex, multifunctional enzymes that synthesize structurally diverse and medicinally important natural products. Given their modular organization, the manipulation of type I PKSs holds tremendous promise for the generation of novel compounds that are not easily accessible by standard synthetic chemical approaches. In theory, hybrid polyketide synthetic pathways can be constructed through the rational recombination of catalytic domains or modules from a variety of PKS systems; however, the general success of this strategy has been elusive, largely due to a poor understanding of the interactions between catalytic domains, as well as PKS modules. Over the past several years, a fundamental knowledge of these issues, and others, has begun to emerge, offering refined strategies for the facile engineering of hybrid polyketide pathways.

Bacterial Physiological Phenomena↗

PubMed2005

Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067.

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.

Ascomycota↗

PubMed2006

Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes.

Fungal polyketides comprise a diverse group of secondary metabolites that play an important role for drug discovery, as pigments, and as mycotoxins. Their biosynthesis is governed by multidomain enzymes, so-called fungal type I polyketide synthases (PKS). Investigating the molecular basis of polyketide biosynthesis in fungi is of great importance for ecological and pharmacological reasons. In addition, cloning, functional analysis and expression of fungal PKS genes also set the basis for engineering the yet largely untapped biosynthetic potential.

Fungal Proteins↗

PubMed2005

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis.

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.

Animals↗

PubMed2006

The structure of a ketoreductase determines the organization of the beta-carbon processing enzymes of modular polyketide synthases.

The structure of the ketoreductase (KR) from the first module of the erythromycin synthase with NADPH bound was solved to 1.79 A resolution. The 51 kDa domain has two subdomains, each similar to a short-chain dehydrogenase/reductase (SDR) monomer. One subdomain has a truncated Rossmann fold and serves a purely structural role stabilizing the other subdomain, which catalyzes the reduction of the beta-carbonyl of a polyketide and possibly the epimerization of an alpha-substituent. The structure enabled us to define the domain boundaries of KR, the dehydratase (DH), and the enoylreductase (ER). It also constrains the three-dimensional organization of these domains within a module, revealing that KR does not make dimeric contacts across the 2-fold axis of the module. The quaternary structure elucidates how substrates are shuttled between the active sites of polyketide synthases (PKSs), as well as related fatty acid synthases (FASs), and suggests how domains can be swapped to make hybrid synthases that produce novel polyketides.

Alcohol Oxidoreductases↗

PubMed2005

Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry.

Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.

Acyl Carrier Protein↗

PubMed2005

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

Acyl Carrier Protein↗

PubMed1997

Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2).

The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.

Acyl Carrier Protein↗