Phylogenetically Diverse Cultivable Fungal Community and Polyketide Synthase (PKS), Non-ribosomal Peptide Synthase (NRPS) Genes Associated with the South China Sea Sponges

Compared with sponge-associated bacteria, the phylogenetic diversity of fungi in sponge and the association of sponge fungi remain largely unknown. Meanwhile, no detection of polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) genes in sponge-associated fungi has been attempted. In this study, diverse and novel cultivable fungi including 10 genera (Aspergillus, Ascomycete, Fusarium, Isaria, Penicillium, Plectosphaerella, Pseudonectria, Simplicillium, Trichoderma, and Volutella) in four orders (Eurotiales, Hypocreales, Microascales, and Phyllachorales) of phylum Ascomycota were isolated from 10 species marine sponges in the South China Sea. Eurotiales and Hypocreales fungi were suggested as sponge generalists. The predominant isolates were Penicillium and Aspergillus in Eurotiales followed by Volutella in Hypocreales. Based on the conserved Beta-ketosynthase of PKS and A domain of NRPS, 15 polyketide synthases, and four non-ribosomal peptides synthesis genes, including non-reducing and reducing PKSs and hybrid PKS–NRPS, were detected in these fungal isolates. A lateral gene transfer event was indicated in the comparison between the phylogenetic diversity of 18S rRNA genes and β-ketoacyl synthase domain sequences. Some fungi, especially those with PKS or NRPS genes, showed antimicrobial activity against P. fluorescens, S. aureus and B. subtilis. It was the first time to investigate PKS and NRPS genes in sponge-associated fungi. Based on the detected antibiotics biosynthesis-related PKS and NRPS genes and antimicrobial activity, the potential ecological role of sponge-associated fungi in the chemical defense for sponge host was suggested. This study extended our knowledge of sponge-associated fungal phylogenetic diversity and their potential roles in the chemical defense.     

Chemical investigation of novel ascomycetes using PCR based screening approaches

Fungi are well known for a wealth of pharmacologically important activities and agrochemical properties. Polyketides that are widely found in fungi, are a large group of secondary metabolites which exhibit diversity in their function and structure. Here we described an investigation of three fungal strains which were prospected for production of polyketides. The aim of this work was to employ the diversity of reducing type I polyketide synthase genes in these fungi using a molecular and bioinformatics approaches as a mini tool. A degenerate primer pair for highly reduced PKSs was newly designed and used together with ketosynthase primers for amplification. One hundred and thirty-eight clones were sequenced. Ten KS domain sequences were isolated, using two primer pairs specific for highly reduced type PKSs. This study revealed four sequences from Emarcea castanopsidicola, four ketosynthase sequences from Gaeumannomyces amomi and two sequences from Leiosphaerella amomi, respectively. Bioinformatic techniques were employed to identify a group of these KS domain sequences. Based on these sequences suggested that rapid screening provided the potential to explore significant PKS structural diversity. Hence chemical investigation had been conducted and exhibited nine compounds. The endophytic fungus L. amomi was cultivated and elucidated linoleic acid, ergosterol and an unidentified sterol in the extracts. Linoleic acid, sitosterol, and p-hydroxybenzoic acid were isolated from the saprobic fungus E. castanopsidicola. We first isolated a new polyketide, stemphol 1-O-β-D-galactopyranoside together with four known metabolites; stemphol, kojic acid, ergosterol, indole-3-carboxylic acid from an ethyl acetate extract of the cultures of G. amomi. Stemphol was classified as a phenolic lipid or resorcinolic lipid, which have biopharmacological, biomedical, and biotechnological importance. However, recent researches have revealed that these molecule types are synthesized by 2′-oxoalkylresorcinolic acid synthase. The prospective KS domain sequences from this study will be used as probes to isolate putative PKS genes. A gene cluster responsible for PK biosynthesis should be confirmed by determination of PK products generated by these enzymes.     

Exploring the diversity of marine-derived fungal polyketide synthases

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.     

Evolution of ketosynthase domains of polyketide synthase genes in the Cladonia chlorophaea species complex (Cladoniaceae)

Lichen-forming fungi synthesize a diversity of polyketides, but only a few non-reducing polyketide synthase (PKS) genes from a lichen-forming fungus have been linked with a specific polyketide. While it is a challenge to link the large number of PKS paralogs in fungi with specific products, it might be expected that the PKS paralogs from closely related species would be similar because of recent evolutionary divergence. The objectives of this study were to reconstruct a PKS gene phylogeny of the Cladonia chlorophaea species complex based on the ketosynthase domain, a species phylogeny of the complex, and to explore the presence of PKS gene paralogs among members of the species complex. DNA was isolated from 51 individuals of C. chlorophaea and allies to screen for the presence of 13 PKS paralogs. A 128 sequence PKS gene phylogeny using deduced amino acid sequences estimated from the 13 PKS paralogs and sequences subjected to BLASTx comparisons showed losses of each of two PKS domains (reducing and methylation). This research provided insight into the evolution of PKS genes in the C. chlorophaea group, species evolution in the group, and it identified potential directions for further investigation of polyketide synthesis in the C. chlorophaea species complex.     

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.     

Diversity of Polyketide Synthase (PKS) genes in metagenomic community of freshwater sponge Lubomirskia baicalensis

The screening of metagenomic DNA of the microbial community associated with the Baikalian sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase (PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active substances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35?C65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various groups of microorganisms, i.e., Alpha-, Beta-, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria; and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A (CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycin (CrpB). The homology of the found sequences with those of the EMBL database lies in the range of 50?C82%, which indicates that the freshwater sponge community contains genes that encode new, not yet studied polyketide substances of potential biotechnological significance.     

Genome screening for reducing type I polyketide synthase genes in tropical fungi associated with medicinal plants

The aim of this work was to employ primers, which encode ketosynthase (KS) domains designed to detect Lovastatin-type PKSs (highly reduced molecules), to identify fungal species that have the potential for polyketide production. Using this strategy we have identified twenty-three KS sequences from twenty different fungal strains associated with medicinal plants found in Thailand. Phylogenetic analysis based on these sequences suggested that rapid screening provided the potential to explore significant PKS structural diversity. With this primer set a unique subclade of reducing type I PKS was identified. This encodes uncharacterized functional enzyme systems, which may suggest a novel function for these pks. Two fungi, Eupenicillium shearii and Myrothecium pandanicola within this novel clade, were investigated for polyketide synthesis. Three compounds, p-hydroxyphenopyrrozin (1) phenopyrrozin (2), and 2,3-dihydro-5-methoxy-2-methylchromen-4-one (3), were identified.     

6-MSAS-like polyketide synthase genes occur in lichenized ascomycetes

Lichenized and non-lichenized filamentous ascomycetes produce a great variety of polyketide secondary metabolites. Some polyketide synthase (PKS) genes from non-lichenized fungi have been characterized, but the function of PKS genes from lichenized species remains unknown. Phylogenetic analysis of keto synthase (KS) domains allows prediction of the presence or absence of particular domains in the PKS gene. In the current study we screened genomic DNA from lichenized fungi for the presence of non-reducing and 6-methylsalicylic acid synthase (6-MSAS)-type PKS genes. We developed new degenerate primers in the acyl transferase (AT) region to amplify a PKS fragment spanning most of the KS region, the entire linker between KS and AT, and half of the AT region. Phylogenetic analysis shows that lichenized taxa possess PKS genes of the 6-MSAS-type. The extended alignment confirms overall phylogenetic relationships between fungal non-reducing, 6-MSAS-type and bacterial type I PKS genes.     

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     

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.     

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.     

Polyketide synthase genes and the natural products potential of Dictyostelium discoideum

MOTIVATION: The genome of the social amoeba Dictyostelium discoideum contains an unusually large number of polyketide synthase (PKS) genes. An analysis of the genes is a first step towards understanding the biological roles of their products and exploiting novel products. RESULTS: A total of 45 Type I iterative PKS genes were found, 5 of which are probably pseudogenes. Catalytic domains that are homologous with known PKS sequences as well as possible novel domains were identified. The genes often occurred in clusters of 2-5 genes, where members of the cluster had very similar sequences. The D.discoideum PKS genes formed a clade distinct from fungal and bacterial genes. All nine genes examined by RT-PCR were expressed, although at different developmental stages. The promoters of PKS genes were much more divergent than the structural genes, although we have identified motifs that are unique to some PKS gene promoters.     

A resource for the in silico identification of fungal polyketide synthases from predicted fungal proteomes

The goal of this study was to develop a tool specifically designed to identify iterative polyketide synthases (iPKSs) from predicted fungal proteomes. A fungi-based PKS prediction model, specifically for fungal iPKSs, was developed using profile hidden Markov models (pHMMs) based on two essential iPKS domains, the β-ketoacyl synthase (KS) domain and acyltransferase (AT) domain, derived from fungal iPKSs. This fungi-based PKS prediction model was initially tested on the well-annotated proteome of Fusarium graminearum, identifying 15 iPKSs that matched previous predictions and gene disruption studies. These fungi-based pHMMs were subsequently applied to the predicted fungal proteomes of Alternaria brassicicola, Fusarium oxysporum f.sp. lycopersici, Verticillium albo-atrum and Verticillium dahliae. The iPKSs predicted were compared against those predicted by the currently available mixed-kingdom PKS models that include both bacterial and fungal sequences. These mixed-kingdom models have been proven previously by others to be better in predicting true iPKSs from non-iPKSs compared with other available models (e.g. Pfam and TIGRFAM). The fungi-based model was found to perform significantly better on fungal proteomes than the mixed-kingdom PKS model in accuracy, sensitivity, specificity and precision. In addition, the model was capable of predicting the reducing nature of fungal iPKSs by comparison of the bit scores obtained from two separate reducing and nonreducing pHMMs for each domain, which was confirmed by phylogenetic analysis of the KS domain. Biological confirmation of the predictions was obtained by polymerase chain reaction (PCR) amplification of the KS and AT domains of predicted iPKSs from V. dahliae using domain-specific primers and genomic DNA, followed by sequencing of the PCR products. It is expected that the fungi-based PKS model will prove to be a useful tool for the identification and annotation of fungal PKSs from predicted proteomes.     

Insect-Specific Polyketide Synthases (PKSs), Potential PKS-Nonribosomal Peptide Synthetase Hybrids,and Novel PKS Clades in Tropical Fungi

Polyketides draw much attention because of their potential use in pharmaceutical and biotechnological applications. This study identifies an abundant pool of polyketide synthase (PKS) genes from local isolates of tropical fungi found in Thailand in three different ecological niches: insect pathogens, marine inhabitants, and lichen mutualists. We detected 149 PKS genes from 48 fungi using PCR with PKS-specific degenerate primers. We identified and classified 283 additional PKS genes from 13 fungal genomes. Phylogenetic analysis of all these PKS sequences the comprising ketosynthase (KS) conserved region and the KS-acyltransferase interdomain region yielded results very similar to those for phylogenies of the KS domain and suggested a number of remarkable points. (i) Twelve PKS genes amplified from 12 different insect-pathogenic fungi form a tight cluster, although along with two PKS genes extracted from genomes of Aspergillus niger and Aspergillus terreus, in reducing clade III. Some of these insect-specific fungal PKSs are nearly identical. (ii) We identified 38 new PKS-nonribosomal peptide synthetase hybrid genes in reducing clade II. (iii) Four distinct clades were discovered with more than 75% bootstrap support. We propose to designate the novel clade D1 with 100% bootstrap support “reducing clade V.” The newly cloned PKS genes from these tropical fungi should provide useful and diverse genetic resources for future research on the characterization of polyketide compounds synthesized by these enzymes.One hallmark of tropical countries is the tremendous availability and diversity of natural resources. Tropical forests, freshwater reservoirs, and seas are home to an uncountable number of species, ranging from microorganisms (e.g., bacteria, fungi, and protozoa) to invertebrates to vertebrates to plants. Thailand is no exception. The country has a large collection of fungi found in different niches and habitats in its ecosystems. Interesting groups include fungi that are associated with insects, those that inhabit the sea, and those that are in lichen complexes; these are referred to here as insect fungi, marine fungi, and lichenized fungi, respectively. The first group is of particular interest because it represents a remarkable relationship (in this case, pathogenesis) between the fungi and their insect hosts. These entomopathogenic fungi were isolated from the dead insect bodies in different stages (e.g., larvae, pupae, nymphs, or adults). The marine fungi used in this study were mostly isolated from the living or dead plant parts floating at the seashore, whereas the lichen mutualistic fungi were isolated from lichen complexes on the bark of trees in tropical forests in Thailand. All these fungal isolates were deposited in National Center for Genetic Engineering and Biotechnology (BIOTEC) Culture Collection (BCC). The BCC has one of the richest collections (approximately 400 species and 5,000 isolates) of insect fungi in the world (19).Secondary metabolites may play an important role in organisms that synthesize them, for example, in spore development (7), protection, or host virulence (5). Polyketides (PKs) are natural secondary metabolite compounds derived from the condensation of acyl coenzyme A subunits in a head-to-tail manner, and they have a tremendous diversity in structure (33). Structural diversification of the PKs includes a variation in the number of subunits, types of subunits, degree of chemical reduction of the β-keto thioester, extent of stereochemistry of the α-keto group at each condensation, and subsequent processing (e.g., cyclicization) (25, 28, 33). The high therapeutic and economic value of PK compounds has attracted the interest of drug companies and government research agencies. Some PKs are commercially available for medical treatments, such as grahamimycin and patulin (antibiotics), lovastatin and compactin (cholesterol-lowering agents), griseofulvin (an antibiotic/antifungal agent), and monocerin (an antifungal agent).Enzymes that synthesize the PKs are called PK synthases (PKSs). PKSs are multifunctional enzymes that are composed of three principal domains: ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP). Fungal PKSs are type I, multifunctional large enzymes and use an iterative strategy to synthesize PKs. They can be divided into two groups, nonreducing (NR) and reducing (4), and further subdivided into NR subclades I, II, and III and reducing subclades I, II, III, and IV (26). NR PKSs include those synthesizing pigments or aflatoxin. Reducing PKSs are involved in the synthesis of PK compounds with various chemical reductions in structure. Apart from the three major domains (KS, AT, and ACP) present in all PKSs, reducing PKSs contain three additional domains, i.e., dehydratase, enoyl reductase, and ketoreductase, which are involved in the reduction of the keto group to various stages (i.e., alcohol, unsaturated thiolester, and full saturation, respectively), therefore enhancing diversity of the PK structure.Kroken et al. (26) studied putative amino acid sequences of the PKS genes previously characterized in fungi and the PKS genes discovered from the genome sequencing projects for eight fungal species in the Ascomycota. PKS genes were found only in the genomes of the Pezizomycotina and not in the sequenced genomes of either Ascomycota in the Taphrinomycota or Saccharomycotina or Basidiomycota in the Hymenomycetes. Thus, we focused our search on the fungi in this subphylum. We aimed to mine valuable PKS genes from this fungal resource. One of the main objectives is to find novel secondary metabolites useful for medical or agricultural applications. One highly regarded example is the “vegetable caterpillar,” where the fungus Cordyceps sinensis grows on Hepialidae caterpillars. The fungus has long been used in traditional Chinese medicine. Extracts of Cordyceps sinensis were reported to have a variety of therapeutic effects, for example, antitumor (6), antioxidant (42), and antiaging (24) activities. The C. sinensis-Hepialidae pair is also called the “body snatcher”. This name comes from the fact that the fungus infects and consumes the insect tissue and fills up the insect cavity with its mycelia. Thus, another objective is to find metabolites involved in interaction between fungal pathogens and their insect hosts. Insect pests pose tremendous losses to humans in regard to health issues (vectors of diseases) and economic issues (crop plant losses by insect pathogens and building structure damage by termites). Little was known regarding the roles of PKs in producing fungi on their interaction with insect host. Better understanding of this relationship might have implications for insect control.We conducted our PKS screening using PCR with the degenerate KA series primers (2). In addition to our preliminary PKS screening with these primers in a few fungi (2), the KA series primers were used to clone the reducing PKS gene for radicicol biosynthesis from the fungus Pochonia chlamydosporia, and later its whole biosynthetic cluster was revealed (37). Here, the method and the primers were also proven to be successful in finding rich resources of hidden metabolic pathways for PK biosynthesis from 48 fungi that were isolated in Thailand and, particularly, have no genome sequences determined. In addition, more than 200 PKS genes were identified from our genome analysis of 13 filamentous fungi.     

Ketosynthase Domain Probes Identify Two Subclasses of Fungal Polyketide Synthase Genes

Analysis of fungal polyketide synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketosynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketosynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal polyketide synthase genes.     

Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules: BIOGENESIS OF THE BROAD SPECTRUM ANTIFUNGAL AND ANTI-OOMYCETE COMPOUND,OOCYDIN A

Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties.     

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.     

Ancient Horizontal Gene Transfer from Bacteria Enhances Biosynthetic Capabilities of Fungi

Background

Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive.

Methodology/Principal Findings

We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA)-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication.

Conclusions/Significance

Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.     

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.     

Fungal diversity in the Atacama Desert

Fungi are generally easily dispersed, able to colonise a wide variety of substrata and can tolerate diverse environmental conditions. However, despite these abilities, the diversity of fungi in the Atacama Desert is practically unknown. Most of the resident fungi in desert regions are ubiquitous. Some of them, however, seem to display specific adaptations that enable them to survive under the variety of extreme conditions of these regions, such as high temperature, low availability of water, osmotic stress, desiccation, low availability of nutrients, and exposure to high levels of UV radiation. For these reasons, fungal communities living in the Atacama Desert represent an unknown part of global fungal diversity and, consequently, may be source of new species that could be potential sources for new biotechnological products. In this review, we focus on the current knowledge of the diversity, ecology, adaptive strategies, and biotechnological potential of the fungi reported in the different ecosystems of the Atacama Desert.