A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin.

Race T of the fungal pathogen Cochliobolus heterostrophus is highly virulent toward Texas male sterile (T) maize and differs from its relative, race O, at a locus (Tox1) that is responsible for the production of T-toxin, a family of linear long-chain (C35 to E41) polyketides. In a previous study, the restriction enzyme-mediated integration procedure was used to mutagenize and tag Tox1. Here, we report that the DNA recovered from the insertion site of one mutant encodes a 7.6-kb open reading frame (2530 amino acids) that identifies a multifunctional polyketide synthase (PKS)-encoding gene (PKS1) with six catalytic domains arranged in the following order, starting at the N terminus: beta-ketoacyl synthase, acyltransferase, dehydratase, enoyl reductase, beta-ketoacyl reductase, and acyl carrier protein. PKS1 is interrupted by four apparent introns (74, 57, 49, and 41 bp) and exists in the genome as a single copy surrounded by highly repetitive, A + T-rich DNA. When PKS1 in race T was inactivated by targeted gene disruption, T-toxin production and high virulence were eliminated, indicating that this PKS is required for fungal virulence. Race O strains, which do not produce T-toxin, lack a detectable homolog of PKS1, suggesting that race T may have acquired PKS1 by horizontal transfer of DNA rather than by vertical inheritance from an ancestral strain.     

A decarboxylase encoded at the Cochliobolus heterostrophus translocation-associated Tox1B locus is required for polyketide (T-toxin) biosynthesis and high virulence on T-cytoplasm maize

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to acetoacetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.     

ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H₂O₂. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation.     

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.     

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.     

A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.     

Toxin-Deficient Mutants from a Toxin-Sensitive Transformant of Cochliobolus Heterostrophus

Tox1 is the only genetic element identified which controls production of T-toxin, a linear polyketide involved in the virulence of Cochliobolus heterostrophus to its host plant, corn. Previous attempts to induce toxin-deficient (Tox(-)) mutants, using conventional mutagenesis and screening procedures, have been unsuccessful. As a strategy to enrich for Tox(-) mutants, we constructed a Tox1(+) strain that carried the corn T-urf13 gene (which confers T-toxin sensitivity) fused to a fungal mitochondrial signal sequence; the fusion was under control of the inducible Aspergillus nidulans pelA promoter which, in both A. nidulans and C. heterostrophus, is repressed by glucose and induced by polygalacturonic acid (PGA). We expected that a transformant carrying this construction would be sensitive to its own toxin when the T-urf13 gene was expressed. Indeed, the strain grew normally on medium containing glucose but was inhibited on medium containing PGA. Conidia of this strain were treated with ethylmethanesulfonate and plated on PGA medium. Among 362 survivors, 9 were defective in T-toxin production. Authenticity of each mutant was established by the presence of the transformation vector, proper mating type, and a restiction fragment length polymorphism tightly linked to the Tox1(+) locus. Progeny of each mutant crossed to a Tox1(+) tester segregated 1:1 (for wild type toxin production vs. no or reduced toxin production), indicating a single gene mutation in each case. Progeny of each mutant crossed to a Tox1(-) tester segregated 1 : 1 (for no toxin production vs. no or reduced toxin production) indicating that each mutation mapped at the Tox1 locus. Availability of Tox(-) mutants will permit mapping in the Tox1 region without interference from a known Tox1 linked translocation breakpoint.     

The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.     

Polymorphic Chromosomes Bearing the Tox2 Locus in Cochliobolus carbonum Behave as Homologs during Meiosis

The HTS1 gene in the Tox2 locus of the fungal pathogen Cochliobolus carbonum race 1 is required for synthesis of a host-selective phytotoxin and for increased virulence on susceptible genotypes of maize. The locus is present in race 1 isolates but absent from isolates of the other races, which do not produce the toxin. By pulsed-field gel electrophoresis and Southern analysis with HTS1 sequences and chromosome-specific markers, the HTS1 gene was detected on a 4-Mb chromosome in one group of isolates and on a 2.3-Mb chromosome in another group, which lacked the 4-Mb chromosome. A chromosome-specific marker from C. heterostrophus hybridized to a 2.3-Mb chromosome in non-toxin-producing isolates and in toxin-producing isolates, including those with a 4-Mb chromosome. A marker from C. carbonum hybridized to the 4-Mb chromosome, but in isolates lacking the 4-Mb chromosome, this marker hybridized to a smaller, 2.0-Mb chromosome. Thus, the Tox2 locus is on different chromosomes in different groups of race 1 isolates. Single ascospore progeny from crosses between isolates having HTS1 on different chromosomes were analyzed for toxin-producing ability, virulence, and the presence and chromosomal location of HTS1. All progeny produced HC toxin in culture, incited race 1-type lesions on susceptible maize genotypes, and contained HTS1 sequences, as determined by PCR amplification with gene-specific primers. Analysis of the chromosomal complements of several progeny indicated that they all had only one Tox2-containing chromosome. Thus, despite their differences in size, these chromosomes behave as homologs during meiosis and may have arisen by a translocation.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

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.     

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.     

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     

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.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome.     

Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum.

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin. In crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2- isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsedfield gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2- strain SB114. Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies. Other independent Tox2+ isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2+ -unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2+ isolates studied here. Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosome location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.     

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.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome. Received: 20 April 1998 / Accepted: 21 September 1998     

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.