Type III polyketide synthases in natural product biosynthesis

Polyketides represent an important class of biologically active and structurally diverse compounds in nature. They are synthesized from acyl-coenzyme A substrates by polyketide synthases (PKSs). PKSs are classified into three groups: types I, II, and III. This article introduces recent studies on type III PKSs identified from plants, bacteria, and fungi, and describes the catalytic functions of these enzymes in detail. Plant type III PKSs have been widely studied, as exemplified by chalcone synthase, which plays an important role in the synthesis of plant metabolites. Bacterial type III PKSs fall into five groups, many of which were identified from Streptomyces, a genus that has been well known for its production of bioactive molecules and genetic alterability. Although it was believed that type III PKSs exist exclusively in plants and bacteria, recent fungal genome sequencing projects and biochemical studies revealed the presence of type III PKSs in filamentous fungi, which provides a new chance to study fungal secondary metabolism and synthesize "unnatural" natural products. Type III PKSs have been used for the biosynthesis of novel molecules through precursor-directed and structure-based mutagenesis approaches.     

Evolution of metabolic diversity: Insights from microbial polyketide synthases

Polyketides are a family of complex natural products that are built from simple carboxylic acid building blocks. In microorganisms, the majority of these secondary metabolites are produced by exceptionally large, multifunctional proteins termed polyketide synthases (PKSs). Each unit of a type I PKS assembly line resembles a mammalian type fatty acid synthase (FAS), although certain domains are optionally missing. The evolutionary analysis of microbial PKS has revealed a long joint evolution process of PKSs and FASs. The phylogenomic analysis of modular type I PKSs as the most widespread PKS type in bacteria showed a large impact of gene duplications and gene losses on the evolution of type I PKS in different bacterial groups. The majority of type I PKSs in actinobacteria and cyanobacteria may have evolved from a common ancestor, whereas in proteobacteria most type I PKSs were acquired from other bacterial groups. The modularization of type I PKSs almost unexceptionally started with multiple duplications of a single ancestor module. The repeating modules represent ideal platforms for recombination events that can lead to corresponding changes in the actual chemistry of the products. The analysis of these “natural reprogramming” events of PKSs may assist in the development of concepts for the biocombinatorial design of bioactive compounds.     

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.     

Type III polyketide synthases in lichen mycobionts

Lichenized and non-lichenized fungi produce a wide range of secondary metabolites. So far, type I polyketide synthases (PKSs) are the suggested catalysts for the biosynthesis of lichen compounds. We were interested whether lichen mycobionts also contain type III PKSs, representing a class that was only recently discovered in fungi. With an alignment of known type III CHS-like genes we applied the CODEHOP strategy to design degenerate PCR primers. We further screened available fungal genomes for type III PKS genes and aligned these sequences for a phylogenetic analysis. Type III-like genes from lichen mycobionts are closely related to those known from non-lichenized fungi, but not to those of bacteria and/or plants. We conclude that type III PKS genes are ubiquitous in fungi. They are present in diverse unrelated lichen mycobionts, but their function in lichens is so far unclear.     

Physcomitrella PpORS,Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees,Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2′-oxoalkylresorcinol. Therefore, PpORS is a 2′-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln²¹⁸, Val²⁷⁷, and Ala²⁸⁶) at the putative PpORS active site. Substitution of the Ala²⁸⁶ to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.     

Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.Type III polyketide synthases (PKSs), represented by a plant chalcone synthase (CHS), are the condensing enzymes that catalyze the synthesis of aromatic polyketides in plants, fungi, and bacteria (2). CHS catalyzes the decarboxylative condensation of p-coumaroyl-coenzyme A (p-coumaroyl-CoA), called a starter substrate, with three malonyl-CoAs, called extender substrates, and synthesizing a tetraketide intermediate. The synthesized tetraketide intermediate was cyclized and aromatized by CHS and resulted in naringenin chalcone. Like CHS, most of the type III PKSs catalyze the condensation of a starter substrate with several molecules of an extender substrate and cyclization. There are many type III PKSs that differ in these specificities.Until recently, type III PKSs were discovered only from plants. In 1999, the first bacterial type III PKS, RppA, was discovered. RppA catalyzes the condensation of five malonyl-CoAs to synthesize 1,3,6,8-tetrahydroxynaphthalene, which is a precursor of hexahydroxyperylenequinone melanin in the actinomycete Streptomyces griseus (4). Since then, the genome projects of various bacteria have revealed that type III PKSs are widely distributed in a variety of bacteria. For example, ArsB and ArsC, both of which are type III PKSs in Azotobacter vinelandii, catalyze the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the cyst membrane (5). In S. griseus, the srs operon consisting of srsA, srsB, and srsC is responsible for the synthesis of methylated phenolic lipids derived from alkylresorcinols and alkylpyrones (6). The function of each of the operon members is that SrsA is a type III PKS responsible for the synthesis of phenolic lipids alkylresorcinol and alkylpyrones, SrsB is a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC is a hydroxylase acting on the alkylresorcinol methyl ethers. The phenolic lipids synthesized by the Srs enzymes confer resistance to β-lactam antibiotics (6). Therefore, it is suggested that phenolic lipids play an important role as minor components in the biological membrane in various bacteria. In fact, srsAB- and srsABC-like operons are distributed widely in both gram-positive and -negative bacteria (see Fig. S1 in the supplemental material). However, most of these type III PKSs have not been characterized.Bacillus subtilis is one of the best-characterized gram-positive bacteria. BcsA, which stands for bacterial chalcone synthase, was annotated as a homologue of type III PKS in B. subtilis (3). As described in this paper, however, this annotation needs correction. We renamed the gene bpsA (for Bacillus pyrone synthase). Moreover, the functional unknown gene ypbQ is located next to bpsA. YpbQ, consisting of 168 amino acid residues, contained an isoprenylcysteine carboxyl methyltransferase (ICMT) domain of the ICMT family members, which are unique membrane proteins that are involved in the posttranslational modification of oncogenic proteins (23). Therefore, the bpsA and ypbQ genes were predicted to form an operon, just like srsA and srsB in the srs operon in S. griseus. We therefore named ypbQ, a thus-far functionally unknown gene, bpsB.In this study, we characterized the functions of BpsA and BpsB by in vivo and in vitro experiments. The in vivo experiments revealed that the overexpression of bpsA in B. subtilis led to the production of triketide pyrones, and the co-overexpression of bpsA and bpsB led to the production of triketide pyrone methyl ethers. The in vitro analysis showed that BpsA produced triketide pyrones and a small amount of tetraketide pyrones and tetraketide resorcinols from long-chain fatty acyl CoA thioesters as starter substrates and malonyl-CoA as an extender substrate. Therefore, BpsA is a type III PKS that is responsible for the synthesis of alkylpyrones, and BpsB is a methyltransferase that acts on the alkylpyrones to yield alkylpyrone methyl ethers. BpsB is the first enzyme found to methylate alkylpyrones. Furthermore, we attempted to analyze the biological function of the aliphatic polyketides by disrupting the bpsA and bpsB genes, but no distinct phenotypic changes were detected under laboratory conditions.     

Molecular Cloning,Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from <Emphasis Type="Italic">Sargassum binderi</Emphasis> (Phaeophyta)

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C₆–C₁₄) to produce tri- and tetraketide pyrones. Mutations at H³³¹ and N³⁶⁴ caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His²²⁷ and Leu³⁶⁶ play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.     

A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites

The superfamily of plant and bacterial type III polyketide synthases (PKSs) produces diverse metabolites with distinct biological functions. PKS18, a type III PKS from Mycobacterium tuberculosis, displays an unusual broad specificity for aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20)) to produce tri- and tetraketide pyrones. The crystal structure of PKS18 reveals a 20 A substrate binding tunnel, hitherto unidentified in this superfamily of enzymes. This remarkable tunnel extends from the active site to the surface of the protein and is primarily generated by subtle changes of backbone dihedral angles in the core of the protein. Mutagenic studies combined with structure determination provide molecular insights into the structural elements that contribute to the chain length specificity of the enzyme. This first bacterial type III PKS structure underlines a fascinating example of the way in which subtle changes in protein architecture can generate metabolite diversity in nature.     

植物Ⅲ型聚酮合酶的分子机制与应用前景

植物III型聚酮合酶能催化生成一系列结构各异、具有不同生理活性、包含查耳酮合酶基本骨架的植物次生代谢产物,这类次生代谢产物不仅使植物体本身的抗逆性提高,并且对人类健康医疗有很好的应用前景。以下综述了近年来从植物中克隆、鉴定III型聚酮合酶的研究进展,着重论述了其分子结构、催化反应的类型和机制、表达调控及其在转基因工程方面的研究和应用前景。这些研究将为有效地对其进行基因改造,合成一些难以化学合成的新型天然化合物奠定基础,并且为将来进一步开展III型聚酮合酶的转基因工程提供了参考。     

Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.     

Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.     

Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning,functional expression,and site-directed mutagenesis of two polyketide synthases

Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.     

Polyketide Synthase Gene Diversity within the Microbiome of the Sponge Arenosclera brasiliensis,Endemic to the Southern Atlantic Ocean

Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase,PKSIII<Emphasis Type="Italic">Nc</Emphasis> in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.     

Widening the bottleneck: Heterologous expression,purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.     

Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells

Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells.     

Unusual intron in the second exon of a Type III polyketide synthase gene of Alpinia calcarata Rosc

Plant phenolic compounds form a valuable resource of secondary metabolites having a broad spectrum of biological activities. Type III polyketide synthases play a key role in the formation of basic structural skeleton of the phenolic compounds. As a group of medicinal plants, PKSs with novel features are expected in the genome of Zingiberaceae. The genomic exploration of PKS in Alpinia calcarata conducted in this study identified the presence of an unusual intron at the region forming the second exon of typical PKSs, forming a gateway information of distribution of novel PKSs in Zingiberaceae.     

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.     

Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia

Bacterial type I polyketide synthases (PKSs) produce a wide range of biomedically important secondary metabolites. These enzymes possess a modular structure that can be genetically re-engineered to yield novel drug candidates not found in nature. Recently, we have reported the putative pederin PKS from an uncultured bacterial symbiont of Paederus fuscipes beetles. It belongs to an architecturally unusual PKS group, the members of which contain iteratively acting acyltransferases that are not integrated into the PKS modules but are encoded by isolated genes. As these systems are rare, often contain additional unusual features and are of smaller size than regular PKSs, the development of a method for the targeted isolation of new group members would be of great interest. Here, we present a phylogenetic approach to identify these systems rapidly in highly complex metagenomic DNA samples. To demonstrate its practical value, we located two pederin-type PKS systems putatively involved in the biosynthesis of antitumour polyketides in the metagenomic DNA of beetles, sponges and their uncultivated bacterial symbionts.