Polyketides in insects: ecological role of these widespread chemicals and evolutionary aspects of their biogenesis

Polyketides are known to be used by insects for pheromone communication and defence against enemies. Although in microorganisms (fungi, bacteria) and plants polyketide biogenesis is known to be catalysed by polyketide synthases (PKS), no insect PKS involved in biosynthesis of pheromones or defensive compounds have yet been found. Polyketides detected in insects may also be biosynthesized by endosymbionts. From a chemical perspective, polyketide biogenesis involves the formation of a polyketide chain using carboxylic acids as precursors. Fatty acid biosynthesis also requires carboxylic acids as precursors, but utilizes fatty acid synthases (FAS) to catalyse this process. In the present review, studies of the biosynthesis of insect polyketides applying labelled carboxylic acids as precursors are outlined to exemplify chemical approaches used to elucidate insect polyketide formation. However, since compounds biosynthesised by FAS may use the same precursors, it still remains unclear whether the structures that are formed from e.g. acetate chains (acetogenins) or propanoate chains (propanogenins) are PKS or FAS products. A critical comparison of PKS and FAS architectures and activities supports the hypothesis of a common evolutionary origin of these enzyme complexes and highlights why PKS can catalyse the biosynthesis of much more complex products than can FAS. Finally, we summarise knowledge which might assist researchers in designing approaches for the detection of insect PKS genes.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.     

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.     

Novel polyketide synthase from Nectria haematococca

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: beta-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.     

Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases

Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.     

Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.     

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398. Received 14 June 2001/ Accepted in revised form 15 July 2001     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

Rational elimination of Aspergillus terreus sulochrin production

Elimination of undesirable co-metabolites from industrial fermentations is often required due to the toxicities associated with the contaminants and/or due to difficulties in removing the contaminants during downstream processing. Sulochrin is a co-metabolite produced during the Aspergillus terreus lovastatin fermentation. Examination of the sulochrin biosynthetic pathway identifies the emodin anthrone polyketide synthase (PKS) at the origin. Thus, genetically disrupting the emodin anthrone PKS gene was expected to result in the elimination of sulochrin biosynthesis. To perform the disruption by homologous recombination, a fragment of the emodin anthrone PKS gene first needed to be isolated. Analysis of several reported fungal PKS amino acid sequences has identified three subfamilies of related sequences (called the Patulin subfamily, the Pigment subfamily, and the Reduction subfamily). PCR primers specific for the Pigment subfamily (of which the emodin anthrone PKS is expected to belong) were used to isolate a fragment of a novel PKS gene from A. terreus. Targeted gene disruption identifies the novel gene fragment as that from the emodin anthrone PKS. Consequently, the gene disruption event eliminated the production of metabolites from the sulochrin biosynthetic pathway.     

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.     

Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.     

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.