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PubMed2003

Polyketide chain length control by chain length factor.

Bacterial aromatic polyketides are pharmacologically important natural products. A critical parameter that dictates product structure is the carbon chain length of the polyketide backbone. Systematic manipulation of polyketide chain length represents a major unmet challenge in natural product biosynthesis. Polyketide chain elongation is catalyzed by a heterodimeric ketosynthase. In contrast to homodimeric ketosynthases found in fatty acid synthases, the active site cysteine is absent from the one subunit of this heterodimer. The precise role of this catalytically silent subunit has been debated over the past decade. We demonstrate here that this subunit is the primary determinant of polyketide chain length, thereby validating its designation as chain length factor. Using structure-based mutagenesis, we identified key residues in the chain length factor that could be manipulated to convert an octaketide synthase into a decaketide synthase and vice versa. These results should lead to novel strategies for the engineered biosynthesis of hitherto unidentified polyketide scaffolds.

Amino Acid Sequence↗

PubMed1996

Engineered biosynthesis of novel polyketides: properties of the whiE aromatase/cyclase.

The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.

Anti-Bacterial Agents↗

PubMed2001

Biosynthesis of polyketides in heterologous hosts.

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.

Biotechnology↗

PubMed1999

Tolerance and specificity of polyketide synthases.

Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Additional complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the molecular recognition features of polyketide synthases. Within this decade, a variety of genetic, biochemical, and chemical investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.

Multienzyme Complexes↗

PubMed1999

Constructing polyketides: from collie to combinatorial biosynthesis.

In a new golden age, polyketides are investigated and manipulated with the tools of molecular biology and genetics; hybrid polyketides can be produced. Pharmaceutical companies hope to find new and useful polyketide products, including antibiotics, anthelminthics, and immunosuppressants. This review describes the past developments (largely chemical) on which the present investigations are based, attempts to make sense of the expanding scope of polyketides, looks at the shifting research focus around polyketides, presents a working definition in biosynthetic terms, and takes note of recent work in combinatorial biosynthesis. Also discussed is the failure of the classical enzymological approach to polyketide biosynthesis.

Anti-Bacterial Agents↗

PubMed1999

Disruption of an aromatase/cyclase from the oxytetracycline gene cluster of Streptomyces rimosus results in production of novel polyketides with shorter chain lengths.

Oxytetracycline is a polyketide antibiotic made by Streptomyces rimosus. From DNA sequencing, the gene product of otcD1 is deduced to function as a bifunctional cyclase/aromatase involved in ring closure of the polyketide backbone. Although otcD1 is contiguous with the ketoreductase gene, they are located an unusually large distance from the genes encoding the "minimal polyketide synthase" of the oxytetracycline gene cluster. A recombinant, disrupted in the genomic copy of otcD1, made four novel polyketides, all of shorter chain length (by up to 10 carbons) than oxytetracycline. All four novel structures contained the unusual carboxamido group, typical of oxytetracycline. This implies that the carboxamido group is present at the start of biosynthesis of oxytetracycline, a topic that has been debated in the literature. Loss of the cyclase protein has a profound influence on the length of polyketide chain assembled, implying that OtcD1 plays a greater role in the overall integrity of the quaternary structure of the polyketide complex than hitherto imagined.

Amino Acid Sequence↗

PubMed2006

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides.

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product's structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.

Amino Acid Sequence↗

PubMed1997

Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase.

The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

Acyl Carrier Protein↗

PubMed2000

Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer.

Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed2006

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units.

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

Acyl Carrier Protein↗

PubMed2001

A biosynthetic classification of fungal and streptomycete fused-ring aromatic polyketides.

Polyketides are one of the largest and most structurally diverse classes of naturally occurring compounds, ranging from simple aromatic metabolites to complex macrocyclic lactones. Fungi and filamentous bacteria, particularly the actinomycetes, are major sources of polycyclic aromatic structures, which include many clinically important antibiotics and other useful metabolites. These fused-ring polyketides are formed by the action of polyketide synthases (PKSs), which catalyse the assembly, folding and cross-linking of poly-beta-ketoacyl intermediates. In view of the taxonomic gulf between the eukaryotic fungi and prokaryotic bacteria, it is not surprising that they are rarely found to produce structurally identical fused-ring metabolites. A review of [(13)C(2)]acetate incorporation data has revealed consistent differences in the reported cyclisation patterns, which require regiospecifically distinct cross-linking of otherwise identical linear polyketide precursors. This observation provides the basis for a structural and biosynthetic classification of microbial fused-ring polyketides, which has a number of useful ramifications.

Anti-Bacterial Agents↗

PubMedDate not supplied

United States Department of Agriculture-Agricultural Research Service studies on polyketide toxins of Fusarium oxysporum f sp vasinfectum: potential targets for disease control.

A group of 133 isolates of the cotton wilt pathogen Fusarium oxysporum Schlecht f sp vasinfectum (Atk) Sny & Hans, representing five races and 20 vegetative compatibility groups within race 1 were used to determine the identity, biosynthetic regulation and taxonomic distribution of polyketide toxins produced by this pathogen. All isolates of F oxysporum f sp vasinfectum produced and secreted the nonaketide naphthazarin quinones, bikaverin and norbikaverin. Most isolates of race 1 (previously denoted as races 1, 2 and 6; and also called race A) also synthesized the heptaketide naphthoquinones, nectriafurone, anhydrofusarubin lactol and 5-O-methyljavanicin. Nine avirulent isolates of F oxysporum from Upland cotton roots, three isolates of race 3 of F oxysporum f sp vasinfectum, and four isolates of F oxysporum f sp vasinfectum from Australia, all of which previously failed to cause disease of Upland cotton (Gossypium hirsutum L) in stem-puncture assays, also failed to synthesize or secrete more than trace amounts of the heptaketide compounds. These results indicate that the heptaketides may have a unique role in the virulence of race 1 to Upland cotton. The synthesis of all polyketide toxins by ATCC isolate 24908 of F oxysporum f sp vasinfectum was regulated by pH, carbon/nitrogen ratios, and availability of calcium in media. Synthesis was greatest below pH 7.0 and increased progressively as carbon/nitrogen ratios were increased by decreasing the amounts of nitrogen added to media. The nonaketides were the major polyketides accumulated in synthetic media at pH 4.5 and below, whereas the heptaketides were predominant at pH 5.0 and above. The heptaketides were the major polyketides formed when 10 F oxysporum f sp vasinfectum race 1 isolates were grown on sterilized stems of Fusarium wilt-susceptible cotton cultivars, but these compounds were not produced on sorghum grain cultures. Both groups of polyketide toxins were apparently secreted by F oxysporum f sp vasinfectum, since half of the toxin in 2-day-old shake culture was present in the supernatant. Secretion was enhanced by calcium. Glutamine and glutamic acid inhibited both nonaketide and heptaketide syntheses, even at low nitrogen

Agriculture↗

PubMed2002

Macrotetrolide biosynthesis: a novel type II polyketide synthase.

Polyketide biosynthesis is catalyzed by polyketide synthase (PKS) and three types of bacterial PKS are known to date. Feeding experiments with isotope-labeled precursors established the polyketide origin of the macrotetrolides, but the labeling pattern cannot be rationalized according to the established PKS paradigm. Genetic analysis of the macrotetrolide biosynthesis unveiled an unprecedented organization for a polyketide gene cluster that features five genes encoding discrete ketoacyl synthase (KS) and four genes encoding discrete ketoreductase (KR) but lacking an acyl carrier protein (ACP). Macrotetrolide biosynthesis is proposed to involve a novel type II PKS that acts directly on acyl CoA substrates, functions noniteratively, and catalyzes both C-C and C-O bond formation. These findings demonstrate once again Nature's versatility in making complex molecules and suggests new strategies for PKS engineering to further expand the scope and diversity of polyketide library. They also should serve as an inspiration in searching for PKS with novel chemistry for combinatorial biosynthesis.

Acyl Coenzyme A↗

PubMedDate not supplied

Polyketide synthase genes from marine dinoflagellates.

Rapidly developing techniques for manipulating the pathways of polyketide biosynthesis at the genomic level have created the demand for new pathways with novel biosynthetic capability. Polyketides derived from dinoflagellates are among the most complex and unique structures identified thus far, yet no studies of the biosynthesis of dinoflagellate-derived polyketides at the genomic level have been reported. Nine strains representing 7 different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKSs) by polymerase chain reaction (PCR) and reverse transcriptase PCR. Seven of the 9 strains yielded products that were homologous with known and putative type I PKSs. Unexpectedly, a PKS gene was amplified from cultures of the dinoflagellate Gymnodinium catenatum, a saxitoxin producer, which is not known to produce a polyketide. In each case the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S ribosomal RNA gene. However, amplification from polyadenylated RNA, the lack of PKS expression in light-deprived cultures, residual phylogenetic signals, resistance to methylation-sensitive restriction enzymes, and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes.

Animals↗

PubMed1999

Polyketides from dinoflagellates: origins, pharmacology and biosynthesis.

Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyketides identified to date. The biological activities of these compounds are quite diverse. Compounds having potential therapeutic value as anti-cancer agents as well as deadly neurotoxins, whose production has resulted in severe public health hazards and economic hardships, are represented in this group of secondary metabolites. Stable isotope feeding experiments have firmly established the polyketide origins of representative compounds from each of the three structural classes, the polyether ladders, the macrocycles and the linear polyethers. Yet some unusual labeling patterns have been observed in each class. Pendant methyl groups are most often derived from C-2 of acetate and deletions of C-1 of acetate are common. Studies on the biosynthesis of dinoflagellate derived polyketides at the genomic level have not been reported, in part due to the peculiarities of the dinoflagellate nucleus and the lack of a dinoflagellate transformation system. Nevertheless, a fundamental understanding of the genetics of polyketide biosynthesis by dinoflagellates could be the catalyst for developing several fruitful avenues of research. Dinoflagellate derived polyketides are reviewed with special emphasis on pharmacology and biosynthesis.

Animals↗

PubMed1999

Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes.

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.

Amino Acid Sequence↗

PubMed2003

Diversity of polyketide synthase gene sequences in Aspergillus species.

Fungal polyketide synthases are responsible for the biosynthesis of several mycotoxins and other secondary metabolites. The aim of our work was to investigate the diversity of polyketide synthases in Aspergillus species using two approaches: PCR amplification using oligonucleotide primers, and bioinformatics. Ketosynthase domain probes amplified DNA fragments of about 700 bp in each examined isolate. Sequences of these domains were aligned and analyzed by phylogenetic methods. The ketosynthase domain sequences were highly diverse indicating that they most probably represent polyketide synthases responsible for different functions. A. albertensis and A. niger ketosynthase domain sequences clustered together with sequences of genes required for pigment biosynthesis (wA) in A. nidulans and P. patulum, while the ketosynthase domain sequence of A. muricatus was most closely related to an A. parasiticus wA type domain sequence, and those of the A. ochraceus isolates formed a distinct clade on the tree. These sequences were highly homologous to an A. terreus naphthopyrone synthase gene. An Aspergillus fumigatus genomic database was also searched for ketosynthase domain sequences, which have been included in the phylogenetic analysis. Altogether 14 putative ketosynthase domain sequences were identified. Clustering of the ketosynthase domain sequences correlated well with the type of metabolites produced by the corresponding polyketide synthases. At least 8 clusters with putative ketosynthase domain sequences of unknown function have been identified. Further studies are in progress to clarify the role of some of the identified polyketide synthase genes.

Amino Acid Sequence↗

PubMed2000

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.

Anti-Bacterial Agents↗