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

Usnea longissima has long been used as a traditional medicine in China, India, Turkey, Canada and Europe. This lichen can produce several bioactive compounds that primarily belong to the polyketide family. The enzymes responsible for the production of these compounds are the polyketide synthases, but the biosynthetic processes in lichens are still unclear. In this study, a cultured mycobiont of Usnea longissima was used to isolate and characterize a polyketide synthase gene (UlPKS1). Complete sequence information regarding UlPKS1 (6,468 bp) was obtained by screening a Fosmid genomic library using a 512-bp fragment corresponding to part of the ketosynthase (KS) domain. Sequence analysis of UlPKS1 suggested that it contained features of a non-reducing fungal type I PKS with a starter unit of ACP transacylase (SAT), ketosynthase (KS), product template (PT), acyl carrier protein (ACP) transacylase, acyltransferase (AT) and thioesterase (TE) domain, and had five intervening introns. The domain organization of UlPKS1 (SAT-KS-AT-PT-ACP-ACP-TE) was quite similar to that of aromatic PKSs, and phylogenetic analysis showed that UlPKS1 belonged to the clade of lichenized fungal non-reducing PKS. RT-PCR analyses revealed that the expression of UlPKS1 was down-regulated by glycine and high concentrations of sorbitol, inositol and fructose and up-regulated by sucrose and glucose. Here, we introduce a non-reducing PKS gene in the lichen-forming fungus U. longissima, with a domain structure similar to the structure of orsellinic acid synthase A (OrsA) which is required for orsellinic acid biosynthesis in Aspergillus nidulans.     

A new reducing polyketide synthase gene from the lichen-forming fungus Cladonia metacorallifera

Lichens produce unique polyketide secondary metabolites including depsides, depsidones, dibenzofurans and depsones. The biosynthesis of these compounds is governed by polyketide synthase (PKS), but the mechanism via which they are produced has remained unclear until now. We reported the 6-methylsalicylic acid synthase (6-MSAS) type of PKS gene, which is a member of the fungal-reducing PKSs. A cultured mycobiont of Cladonia metacorallifera was employed in the isolation and characterization of a polyketide synthase gene (CmPKS1). The complete sequence information for CmPKS1 was acquired via the screening of a Fosmid genomic library with a 456 bp fragment corresponding to part of the acyl transferase (AT) domain as a probe. CmPKS1 contains β-ketoacyl synthase (KS), AT, dehydratase (DH), ketoreductase (KR) and phosphopantetheine attachment site (PP) domains.: The domain organization of CmPKS1 (KS-AT-DH-KR-PP) is a typical 6-MSAS-type PKS, and the results of phylogenetic analysis showed that CmPKS1 grouped with other fungal-reducing PKSs. Quantitative real time PCR analyses showed that CmPKS1 was expressed preferentially in the early growth stage of the axenically cultured mycobiont. Furthermore CmPKS1 expression was found to be dependent on the carbon sources and concentrations in the medium.     

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.     

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.     

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.     

Diversity of non-reducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspective

Lichenized fungi synthesize a great variety of secondary metabolites. These are typically crystalline compounds, which are deposited extracellularly on the fungal hyphae. While we know a lot about the chemical properties and structures of these substances, we have very little information on the molecular background of their biosynthesis. In the current study we analyze the diversity of non-reducing polyketide synthase (PKS) genes in members of the lichenized Pertusariales. This order primarily contains fully oxidized secondary metabolites from different substance classes, and is chemically and phylogenetically well studied. Using a degenerate primer approach with subsequent cloning we detected up to five non-reducing PKS sequences in a single PCR product. Eighty-five new KS sequence fragments were obtained for this study. Analysis of the 157 currently available fungal KS sequence fragments in a Bayesian phylogenetic framework revealed 18 highly supported clades that included only lichenized taxa, only non-lichenized taxa, or both. Some Pertusarialean groupings of PKS sequences corresponded partly to phylogenetic groupings based on ribosomal DNA. This is reasonable, because a correlation between well-supported phylogenetic lineages and the occurrence of secondary metabolites in the Pertusariales has been observed before. However, no clear linkage was found between the PKS genes analyzed and the ability to produce a particular secondary substance. Several PKS clades did not reveal obvious patterns of secondary compound distribution or phylogenetic association. Compared with earlier phylogenetic analyses of KS sequences the increased sampling in the current study allowed us to detect many new groupings within the fungal non-reducing PKSs.     

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.     

Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum)

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.     

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.     

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.     

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.     

Characterization of Streptomyces argillaceus genes encoding a polyketide synthase involved in the biosynthesis of the antitumor mithramycin

Mithramycin (Mtm) is an aromatic polyketide which shows antibacterial and antitumor activity. From a chromosomal cosmid library of Streptomyces argillaceus, a Mtm producer, a clone (cosAR7) was isolated by homology to the actI/III region of S. coelicolor and the strDEM genes of S. griseus. From this clone, a 5.3-kb DNA region was sequenced and found to encode six open reading frames (designated as mtmQXPKSTI), five of them transcribed in the same direction. The deduced products of five of these genes resembled components of type-II polyketide synthases. The mtm genes would code for an aromatase (mtmQ), a polypeptide of unknown function (mtmX), a β-ketoacylsynthase (mtmP) and a related ‘chain length factor’ (mtmK), an acyl carrier protein (mtmS) and a β-ketoreductase (mtmT1). The involvement of this gene cluster in Mtm biosynthesis was demonstrated by the Mtm non-producing phenotype of mutants generated in two independent insertional inactivation experiments.     

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).     

Effects of manganese on the viability of vegetative diaspores of the epiphytic lichen Hypogymnia physodes

Soredia of the lichen Hypogymnia physodes cultivated with Bold's basal medium on agar plates for 8 days exhibited decreasing growth rates along with increasing Mn concentrations above 3 mM. Ca and Mg added separately or in combination, alleviated Mn toxicity. The chlorophyll a and b content of the soredia was reduced under the influence of Mn and was positively correlated with the rate of grown soredia. Trebouxia cells of the soredia grown with excess Mn were smaller than control cells, had reduced chloroplasts and were partly collapsed; fungal hyphae were shortened and strongly swollen. Disintegrated cell walls occurred both in algal and fungal cells. Excess Mn was sequestered in extracellular encrustations together with phosphate as corresponding anion. Intracellularly, Mn was accumulated in polyphosphate granules both in algal and fungal cells. Mn uptake was correlated with significant loss of Na, Mg and Ca, particularly from the mycobiont. Fungal cell walls also lost significant amounts of P. The same damage symptoms occurred in cells of soredia both grown or not, but the former had a higher share of intact cells. Damaged cells of both types of soredia had equally increased Mn concentrations, whereas the total Mn content was higher in not grown soredia than in the grown ones due to the greater amount of damaged cells in the former. The Si–Mn ratio in cell walls of intact Trebouxia cells was significantly higher than in collapsed cells. The experimental evidence of Mn sensitivity of H. physodes soredia corresponds to studies of epiphyte vegetation in montane spruce forests of northern Germany that revealed decreasing cover values of H. physodes with an increasing Mn content of the substrate.