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Appl Environ Microbiol. Jun 2012; 78(12): 4468–4480.
PMCID: PMC3370568

Biosynthesis of Fusarubins Accounts for Pigmentation of Fusarium fujikuroi Perithecia


Fusarium fujikuroi produces a variety of secondary metabolites, of which polyketides form the most diverse group. Among these are the highly pigmented naphthoquinones, which have been shown to possess different functional properties for the fungus. A group of naphthoquinones, polyketides related to fusarubin, were identified in Fusarium spp. more than 60 years ago, but neither the genes responsible for their formation nor their biological function has been discovered to date. In addition, although it is known that the sexual fruiting bodies in which the progeny of the fungus develops are darkly colored by a polyketide synthase (PKS)-derived pigment, the structure of this pigment has never been elucidated. Here we present data that link the fusarubin-type polyketides to a defined gene cluster, which we designate fsr, and demonstrate that the fusarubins are the pigments responsible for the coloration of the perithecia. We studied their regulation and the function of the single genes within the cluster by a combination of gene replacements and overexpression of the PKS-encoding gene, and we present a model for the biosynthetic pathway of the fusarubins based on these data.


The rice-pathogenic ascomycete Fusarium fujikuroi (teleomorph, Gibberella fujikuroi MP-C), belonging to the Gibberella fujikuroi species complex, was first isolated from infected rice plants in 1890 (33) and was identified as the causative agent of “bakanae,” or foolish seedling disease, due to its ability to produce gibberellic acids (GAs) (34, 44). Typical symptoms of this disease are chlorotic, slender, and hyperelongated internodes as well as empty or sterile grains (69). Besides GAs, the fungus produces a variety of other secondary metabolites, such as the red polyketide bikaverin, accompanying the production of terpenoid GAs in liquid culture (10). Secondary metabolite genes are often organized in gene clusters (32), such as those found in F. fujikuroi for the seven GA-biosynthetic genes (7) and the six genes involved in bikaverin biosynthesis (48, 80). It is assumed that spatial proximity facilitates the coregulation of secondary metabolite genes within the cluster via, e.g., epigenetic processes, transcriptional regulation by global regulators, or pathway-specific transcription factors located within a gene cluster (53). Most of the secondary metabolite gene clusters are silent under laboratory conditions (8). Different approaches have been undertaken for the activation of those gene clusters, e.g., optimization of culture conditions (pH, nutrient availability) (6, 81), overexpression of pathway-specific transcription factors (9) or global transcriptional regulators, such as LaeA (53), and histone modifications affecting epigenetic control (68).

Secondary metabolites can be grouped into four different classes depending on their structural properties: polyketides, terpenes, nonribosomal peptides, and amino acid-derived compounds. Among secondary metabolites, polyketides form the most abundant group (37), including most of the green and red fungal pigments, all of which belong to the group of naphthoquinones.

These polyketides are synthesized by multidomain nonreducing polyketide synthases (NR-PKSs). NR-PKSs are the minimal fungal PKSs, containing a β-ketoacyl synthase (KS) domain, a malonyl coenzyme A (malonyl-CoA):acyl carrier protein (ACP) transacylase (MAT) domain, and one or two ACP domains, resulting in a defined carbon skeleton through several rounds of elongation (14). Recently, two new domains were identified by Crawford et al.: the starter unit ACP transacylase (SAT) domain, responsible for selecting the starter unit, and the product template (PT) domain. The SAT domain, which has high similarity to the MAT domain, was shown to accept acyl units other than malonyl-CoA as starter units and is widespread among fungal NR-PKSs (15, 16, 17). The PT domain, located between the acyl transferase (AT) and the ACP domain, is responsible for specific aldol cyclization and aromatization of poly-β-keto species, thereby determining the chain length and specific cyclization patterns of the polyketide precursors in fungal NR-PKSs (17, 18). Finally, the respective polyketide is released through various release mechanisms (24), of which thioesterase (TE)-mediated product release by a canonical TE domain is the most common. This domain often extends to a domain capable of C-C Claisen cyclization (a TE/CLC domain) in fungal NR-PKSs, e.g., in Aspergillus parasiticus PksA (42). In some cases, NR-PKSs contain a reductase-releasing (R) domain; in others, e.g., the PKS involved in asperthecin biosynthesis in Aspergillus nidulans, there is no releasing domain at all (14).

So far, the structures of more than 100 naphthoquinone metabolites have been elucidated (52), indicating the structural diversity of this group. The ability to produce naphthoquinones is widespread among fungal organisms, especially among members of the genus Fusarium. To date, only a few of these compounds could be linked to PKS-encoding gene clusters. Among them are the following red pigments: bikaverin in F. fujikuroi (48, 79), aurofusarin in Fusarium graminearum (28, 39, 51), and the perithecial pigments in Fusarium solani (30), F. graminearum (28), and F. verticillioides (60), whose structures have not been identified yet.

In F. fujikuroi, the only PKS described is the bikaverin-specific NR-PKS Bik1 (79). In this study, we present the identification and functional characterization of a second NR-PKS in F. fujikuroi with homology to two PKSs from other Fusarium spp., responsible for the pigmentation of perithecia: the Pgl1 proteins from F. graminearum (28) and F. verticillioides (60). Targeted deletion of this PKS-encoding gene in two F. fujikuroi strains of opposite mating types, and their subsequent sexual crossing, confirmed its role in perithecial pigmentation in F. fujikuroi as well. Detailed molecular analyses revealed the presence of a gene cluster comprising six coregulated genes. Chemical analyses of the products accumulated by the single deletion mutants of these genes proved this gene cluster to be responsible for the biosynthesis of naphthoquinones with structural similarity to fusarubin and allowed us to establish the biosynthetic pathway. To our knowledge, this is the first report to pinpoint perithecial pigmentation in Fusarium spp. to the gene cluster responsible for the production of fusarubins.


Fungal strains and culture conditions.

The wild-type (Wt) Fusarium fujikuroi strain IMI58289 (Commonwealth Mycological Institute, Kew, United Kingdom) was used as a parent strain for all knockout experiments. As a mating partner, F. fujikuroi C1995 (kindly provided by J. F. Leslie, Kansas State University) was used. For all liquid cultivations, the F. fujikuroi strains were preincubated for 72 h in 300-ml Erlenmeyer flasks with 100 ml Darken medium (DVK) (22) on a rotary shaker at 28°C and 180 rpm. A 500-μl aliquot of this culture was used for inoculation of synthetic ICI (Imperial Chemical Industries Ltd., United Kingdom) medium (29) with either 6 mM glutamine or 6 mM sodium nitrate, and incubation was carried out for an additional 1 to 12 days. For protoplasting, 500 μl of the preincubated culture was taken and transferred to 100 ml ICI medium with 10 g/liter fructose instead of glucose and 1 g/liter (NH4)2SO4 as the nitrogen source; this mixture was incubated on a rotary shaker at 28°C and 180 rpm for no longer than 16 h. For the nitrogen shift experiments, the mycelia were grown in ICI medium on a rotary shaker at 28°C and 180 rpm. After 4 days, either 33 mM glutamine, 33 mM sodium nitrate, or no nitrogen was added, and the cultures were incubated for an additional 30 min. For the identification of the compounds, the fungus was grown for 7 days in ICI medium on a rotary shaker at 28°C and 180 rpm. For pH shift experiments, the strains were grown in ICI medium for 4 days on a rotary shaker at 28°C and 180 rpm; then they were transferred to ICI medium with no nitrogen source, adjusted to a pH of either 4 or 8, for an additional 2 h (11). For sexual crosses, carrot agar was used according to the method of Klittich and Leslie (41). For DNA isolation, the fungus was grown on cellophane sheets on solidified complete medium (CM) for 3 days at 28°C. For RNA isolation, the fungus was grown in ICI medium with the desired nitrogen source for 2 to 7 days at 28°C and 180 rpm after preincubation in DVK.

HPLC-DAD analyses of the naphthoquinones.

For high-performance liquid chromatography (HPLC)-diode array detector (DAD) analysis, culture fluid from 1- to 12-day-old cultures was filtered over a 0.2-μm-pore-size membrane filter (Millex; Millipore) and was used directly without further preparation. The samples were separated on a 150- by 3.00-mm, 3-μm Gemini 3u C6-phenyl 110-Å column (Phenomenex, Aschaffenburg, Germany) by using a binary gradient delivered by a Hitachi L-7100 (Merck) pump with 1% formic acid as solvent A and acetonitrile (ACN) as solvent B. The binary gradient started with a linear gradient from 20% B to 50% B in 15 min, followed by an isocratic step for 5 min and an additional linear gradient step to 75% B in 5 min. After each HPLC run, the column was washed by increasing the gradient to 100% B and was equilibrated for 5 min under the starting conditions. The flow rate was set to 0.3 ml min−1. The desired compounds were detected using a Hitachi L-2455 Elite LaChrom DAD. To compare the chromatograms of different culture filtrates, the wavelength was set to 450 nm.

HPLC-UV-FTMS analyses of the naphthoquinones.

Liquid media of 1- to 12-day-old cultures were filtered over a 0.2-μm-pore-size membrane filter and were directly analyzed by high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (FTMS) detection (HPLC-UV-FTMS). To this end, an Accela LC 60057-60010 system (Thermo Fisher Scientific, Bremen, Germany) was linked to a linear trap quadrupole (LTQ) Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Data were acquired with Xcalibur 2.07 SP1 (Thermo Scientific). The samples were separated on a 150- by 3.00-mm, 3 μm Gemini 3u C6-phenyl 110-Å column (Phenomenex, Aschaffenburg, Germany) by using a binary gradient as described above. The flow rate was set to 0.3 ml min−1. Ionization was performed with heated electrospray ionization. Further mass spectrometer conditions were as follows: capillary temperature, 225°C; atmospheric pressure chemical ionization (APCI) vaporizer temperature, 250°C; sheath gas flow, 40 units; auxiliary gas flow, 20 units; source voltage, 3.5 kV; capillary voltage, 35 V; tube lens, 110 V; multiple 00 offset, −4.00 V; lens 0 voltage, −4.20 V; gate lens offset, −35.00 V; multipole 1 offset, −8.00 V; front lens, −5.25 V. Scan events were as follows. (i) A total-ion scan of a mass range from m/z 250 to 800 with a resolution of 30,000 in the positive-ion mode (FTMS) was carried out. (ii) Depending on scan event i, the most intense parent ion was fragmented in the ion trap mass spectrometry (ITMS). If no parent mass was found, the most intense ion was fragmented: activation type, collision-induced dissociation (CID); normalized collision energy, 35; activation Q, 0.250; isolation width, 2. (iii) Depending on scan event i, the most intense parent ion of the list was fragmented. If no parent mass was found, the most intense ion was fragmented in the FTMS: activation type, higher-energy collisional dissociation (HCD); normalized collision energy, 50; activation Q, 0.250; isolation width, 1.5; resolution, 15,000. (iv) A total-ion scan of a mass range from m/z 200 to 800 in the negative-ion mode with the ITMS was carried out. The exact masses of the compounds were identified if the proton adduct ([M + H]+), and, in addition, the sodium adduct ([M + Na]+) or the exact mass in the negative-ion mode ([M − H]) was also detected.

Purification of the naphthoquinoid metabolites.

For the purification of the naphthoquinones, 1 liter of 7-day-old culture fluid of the wild type (Wt) (for final products), the Δfsr2 and Δfsr3 deletion mutants (for intermediates), and the Δfsr2-5/gpd::fsr1 strain (for the PKS-derived product) was extracted by solid-phase extraction (SPE) using C18 SPE cartridges (Phenomenex, Aschaffenburg, Germany). Further purification was accomplished by semipreparative HPLC on a 250- by 10.00-mm, 5 μm Gemini 5u C6-phenyl 110-Å column (Phenomenex, Aschaffenburg, Germany) by using an isocratic gradient delivered by a Jasco PU-2087 pump with 1% formic acid as solvent A and ACN as solvent B. The flow rate was set to 3 ml min−1. The desired compounds were detected using a Jasco UV-2075 UV detector at 450 nm (for final products) or 400 nm (for intermediates and PKS-derived products). Naphthoquinoid metabolites 1 to 8 were obtained after semipreparative HPLC in amounts of 10 to 30 mg for structure elucidation.

NMR measurements.

1H, 13C, and 2-dimensional (2-D) nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DPX-400 (Bruker BioSpin, Rheinstetten, Germany) NMR spectrometer. Signals are reported in parts per million referenced to dimethyl sulfoxide (DMSO)-d6. For structural elucidation and NMR signal assignment, 2-D NMR experiments, such as (H,H)-correlated spectroscopy (H,H-COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC) experiments, were performed. Pulse programs for these experiments were taken from the software library.

Sequence data and phylogenetic analyses.

BLASTP analysis (1) was performed using the predicted protein sequences of Fsr1 to Fsr6, as well as FF_03983 and FF_03990, against NCBI nonredundant protein sequences (Table 1). PKS protein sequences were retrieved from the following sources: for F. graminearum, F. oxysporum, and F. verticillioides, the Broad Institute (www.broadinstitute.org); for F. solani, the Joint Genome Institute (www.jgi-psf.org); and for characterized PKSs of other fungi (14, 32), NCBI (www.ncbi.nlm.nih.gov). KS domains used for phylogenetic analyses were extracted using the PKS/NRPS Analysis website (http://nrps.igs.umaryland.edu/nrps) (4). Phylogenetic analysis was performed by comparing the KS domains using the Web-based tool at www.phylogeny.fr (23).

Table 1
Information about the putative fsr gene cluster

Plasmid constructions.

The F. fujikuroi knockout strains were created using yeast recombinational cloning (20). The 5′ and 3′ flanks of the corresponding genes were amplified with appropriate primer pairs (see Table S1 in the supplemental material); for the 5′ flank, primers 5F and 5R were used, and for the 3′ flank, primers 3F and 3R were used, based on the genomic sequence of F. fujikuroi IMI58289 (B. Tudzynski and coworkers, unpublished data). The hygromycin resistance cassette, consisting of the hygromycin B phosphotransferase gene hph (31), driven by the trpC promoter, was amplified using the hph-F/hph-R primer pair from the template pCSN44 (66). Alternatively, the nourseothricin resistance cassette, also driven by the trpC promoter, was used. The nourseothricin resistance cassette was amplified by using plasmid pZPnat1 (GenBank accession number AY631958.1) as a template with the primer pair hphF/hphR-trpC-T2. All primers used are listed in Tables S1 to S4 in the supplemental material. The fragments obtained were cloned into the Saccharomyces cerevisiae strain FY834 (82) together with the EcoRI/XhoI-restricted plasmid pRS426 (19). For restoration of pigment biosynthesis in the Δfsr1 mutant, the respective gene was amplified with the corresponding terminator sequence in four PCRs. All four amplified products were cloned into Saccharomyces cerevisiae FY834 together with the HindIII-restricted modified plasmid pRS426, which additionally contained a nourseothricin resistance cassette driven by an oliC promoter amplified from pNR1 (50) using the primer pair nat-OE-prom/nat-OE-term. The gene of interest itself was driven by the gpd promoter, which was amplified from pveAgfp (80) using the gpd-yeast-for/gpd-yeast-rev primer pair.

Standard molecular methods.

For DNA isolation, lyophilized mycelium was ground to a fine powder in liquid nitrogen, dispersed in extraction buffer according to the method of Cenis (12), and afterwards used for PCR amplification and Southern blot analyses. PCR mixtures contained 25 ng genomic DNA, 5 pmol of each primer, 200 nM deoxynucleoside triphosphates, and 1 U of BioTherm DNA polymerase (GeneCraft GmbH, Lüdinghausen, Germany) for diagnostic PCR and amplification of the flanks for yeast recombinational cloning. PCRs were performed with an initial denaturing step at 94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 1 min at 56 to 60°C, and 1 to 2 min at 70°C, and a final elongation step at 70°C for 10 min. For amplification of the knockout constructs, the TaKaRa polymerase kit was used as recommended by the manufacturer.

For restoration of fusarubin biosynthesis in the Δfsr1 mutant, as well as for the identification of the PKS-derived product, fsr1 was amplified in 4 fragments by using a proofreading polymerase. Under these conditions, PCR mixtures contained 25 ng genomic DNA, 5 pmol of each primer, and 1 U of Phusion polymerase (Finnzymes, Thermo Fisher Scientific, Finland).

For Southern blot analysis, genomic DNA was completely digested using appropriate enzymes, separated on a 1% (wt/vol) agarose gel, and transferred to nylon membranes (Nytran SPC; Whatman) by downward blotting (3). 32P-labeled probes were generated using the random oligomer-primer method and were hybridized to the membranes overnight at 65°C according to the protocol of Sambrook et al. (64). After hybridization, the membrane was washed with 1× SSPE (0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7])–0.1% sodium dodecyl sulfate (SDS) at the same temperature.

For RNA isolation, lyophilized mycelium was ground with a mortar and pestle, and the powder was extracted by using the RNAagents total-RNA isolation kit (Promega, Germany) according to the manufacturer's instructions. Twenty micrograms per sample was loaded onto a 1% agarose gel and was run under denaturing conditions (64). The separated RNA was transferred to nylon membranes (Nytran SPC; Whatman). 32P-labeled probes were created as described above and were hybridized to the membrane overnight at 65°C. Plasmid DNA from Saccharomyces cerevisiae was extracted using the yeast plasmid isolation kit (SpeedPrep; Dualsystems Biotech) and was directly used for PCR.

Fungal transformations.

Protoplasts were prepared from F. fujikuroi IMI58289 as described previously (74). About 107 protoplasts were transformed with 10 μg of the amplified replacement cassettes for the knockouts of the putative gene cluster (fsr1 to fsr6) and one gene each upstream (FF_03983) and downstream (FF_03990) of the cluster. For the restoration of fsr1 and for the identification of the PKS-derived product, the fsr1 and Δfsr2-5 deletion mutants were transformed with 10 μg of the pRS426 vector containing fsr1 driven by the gpd promoter.

For sexual crosses, C1995, the mating partner of F. fujikuroi IMI 58289, was also transformed with the knockout cassette for fsr1. Transformed protoplasts were regenerated as described by Tudzynski et al. (75). The medium contained the appropriate resistance marker. Single conidial cultures were established from either hygromycin B- or nourseothricin-resistant transformants and were used for DNA isolation and Southern blot analysis.

Nucleotide sequence accession number.

The nucleotide and protein sequences of the fsr gene cluster have been deposited in GenBank under accession number HE613440.


Modification of culture condition reveals a second red polyketide in Fusarium fujikuroi.

In F. fujikuroi, the biosynthesis of both GAs and bikaverin is strongly repressed by high levels of glutamine. In synthetic ICI medium with low levels of glutamine (6 mM), the fungus starts to produce these metabolites after nitrogen depletion (10, 79). However, when the fungus was grown with 6 mM sodium nitrate as the sole nitrogen source, only GAs were produced; accumulation of bikaverin was no longer detected, due to the alkaline pH (Fig. 1B). Surprisingly, the liquid culture still was deeply red pigmented after approximately 5 days of cultivation (Fig. 1A). High-performance liquid chromatography (HPLC) and HPLC coupled with Fourier transformation mass spectrometry detection (HPLC-UV-FTMS) (data not shown) confirmed that the red pigment was not bikaverin (Fig. 1B). Measurements of the pH values of the different growth media over a time course showed significant differences depending on the nitrogen source. While the pH was maintained at acidic values between 5 and 6 with glutamine, the pH increased to values of 6.5 to 7.5 in cultures with sodium nitrate (Fig. 1C). The ambient alkaline conditions explain the lack of bikaverin biosynthesis, as shown previously (79). The second red pigment, in contrast, was produced only if the ambient pH of the liquid culture immediately increased to a neutral or alkaline value. Thus, pH conditions in the first 24 h after inoculation seem to determine which of the pigments is produced.

Fig 1
Identification of a new group of red pigments by changing standard growth conditions. Wild-type (Wt) F. fujikuroi was grown in ICI medium with either 6 mM glutamine (Gln) or 6 mM sodium nitrate (NO3) for 5 days. Samples were taken and were used ...

Cloning, disruption, and characterization of the responsible polyketide synthase-encoding gene (fsr1).

Due to the intense red color of the cryptic compound, we assumed structural similarity to aromatic pigments synthesized by NR-PKSs, and hence we hypothesized the involvement of a nonreducing PKS in the biosynthesis of this compound. A BLASTP analysis (1) with the Bik1 sequence against the recently sequenced genome of F. fujikuroi IMI58289 (Tudzynski and coworkers, unpublished) revealed a second NR-PKS with 40% identity (E value, 0) to Bik1. This PKS (Fsr1) consists of 2,286 amino acids (aa), encoded by an open reading frame (ORF) of 7,069 bp, which is interrupted by four putative introns. ClustalW alignment of Fsr1 with the well characterized NR-PKSs responsible for aflatoxin production in A. parasiticus (PksA), perithecial pigment production in F. solani (PksN), and bikaverin production in F. fujikuroi (Bik1) revealed the presence of the following domains. (i) In the N terminus, the recently identified SAT domain, harboring the active GXCXG site, can be found (Fig. 2A; see also Fig. S1 in the supplemental material). (ii) The SAT domain is followed by the KS and MAT domains (Fig. 2A; see also Fig. S1). (iii) Next, we identified a domain with high similarity to the newly identified PT domain presenting the catalytic histidine and aspartate residues (Fig. 2A; see also Fig. S1). (iv) Adjacent to the PT domain, Fsr1 possesses two ACP domains (Fig. 2A; see also Fig. S1). (v) At the C terminus, the enzyme exhibits a reductase (R) domain (Fig. 2A) instead of the canonical TE domain. In contrast to TE and TE/CLC domains, R domains show sequence similarities to the short-chain dehydrogenase/reductase (SDR) superfamily, exhibiting Rossman fold structure and nucleotide binding motifs (36). ClustalW alignment of the Fsr1 R domain with characterized R domains from bacterial and fungal PKSs (5, 13, 38, 45) and the R domain of the yeast α-aminoadipate reductase Lys2p (25) shows high amino acid conservation (see Fig. S2 in the supplemental material).

Fig 2
Phylogeny, PKS domains, and the gene cluster. (A) KS domains of fungal PKSs used for the analysis were extracted, and a phylogram was generated as described in Materials and Methods. For characterized PKSs, the leaves of the phylogram are labeled with ...

Phylogenetic analysis of the KS domains of characterized fungal NR-PKSs revealed a close relatedness of Fsr1 to the Pgl1 proteins of F. verticillioides and F. graminearum, both of which have been shown to be responsible for perithecial pigmentation (28, 60). However, the chemical structures of these perithecial pigments have not been elucidated yet.

In order to investigate whether fsr1 is involved in the production of the cryptic pigment observed in axenic culture (Fig. 1B), the ORF was deleted by targeted gene replacement. Three independent knockout mutants were obtained and were designated Δfsr1-T4, Δfsr1-T6, and Δfsr1-T8. Diagnostic PCR (data not shown) and Southern blot analysis (see Fig. S3 in the supplemental material) confirmed the absence both of the wild-type gene and of additional ectopic integrations of the replacement fragment. When grown in a medium with 6 mM sodium nitrate, none of the mutants exhibited red pigmentation, indicating that Fsr1 is responsible for the biosynthesis of the novel red pigment. Since all Δfsr1 mutants behaved similarly (see Fig. S4 in the supplemental material), Δfsr1-T4 was arbitrarily chosen for subsequent work in this study. Reintroduction of the complete ORF into the Δfsr1 mutant resulted in full restoration of pigment biosynthesis, confirming that Fsr1 is indeed responsible for the formation of the new red pigments (Fig. 3C).

Fig 3
fsr1 is responsible for the production of fusarubins. Fungal strains were grown in 6 mM sodium nitrate. Culture filtrates were taken after 6 days of growth, when the wild type (Wt) showed red pigmentation. Samples were used directly for HPLC-DAD measurements, ...

Identification and characterization of the corresponding gene cluster.

Since the biosynthetic genes for many fungal secondary metabolites are organized in gene clusters (32), we analyzed the genes in the proximity of fsr1. The five genes downstream of fsr1 (Fig. 2B) show homologies to genes known to be typically involved in the biosynthesis of secondary metabolites (Table 1). To determine whether these genes adjacent to fsr1 are involved in pigment biosynthesis, we studied their expression profiles under biosynthesis-favoring (6 mM sodium nitrate) and non-biosynthesis-favoring (6 mM glutamine) conditions. Northern blot analyses revealed the coregulation of six genes (fsr1 to fsr6). FF_03983, upstream of fsr1, and FF_03990, downstream of fsr6, are not expressed and therefore are probably not involved in the biosynthesis (Fig. 4).

Fig 4
Coregulation of the putative fsr gene cluster. The wild type (Wt) was grown in ICI medium with either 6 mM glutamine (Gln) or 6 mM sodium nitrate (NO3). Lyophilized mycelium was used for Northern blot analyses. The putative genes of the fsr gene ...

Regulation of the fsr gene cluster.

To gain deeper insight into the regulation of fsr gene expression, a time course growth experiment was performed under pigment-favoring conditions. Northern blot analyses revealed time-dependent expression of the fsr genes (shown for fsr1 to fsr3): transcripts are detectable at the third day; their intensity peaks at the fourth day and then decreases continuously until the signals are almost undetectable at day 6 postinoculation (Fig. 5A). To further unravel the pH-dependent expression, the wild type was grown under biosynthesis-favoring conditions for 5 days before the mycelium was shifted into synthetic ICI medium without any nitrogen source and with the pH value set either to 4 or to 8. The results clearly show that fsr2 and fsr3 gene expression is repressed under acidic conditions, while the genes are expressed under alkaline conditions (Fig. 5B).

Fig 5
The expression of fsr genes depends on culture age, pH, and nitrogen. (A) The wild type (Wt) was grown in ICI medium with 6 mM sodium nitrate. The mycelium was harvested after 2, 3, 4, 5, 6, and 7 days (t/d). Lyophilized mycelium was used for Northern ...

To study the impact of nitrogen quality and quantity on gene expression in more detail, the wild type was grown for 5 days under pigment production conditions. Then either 33 mM glutamine, 33 mM sodium nitrate, or water (no nitrogen) was added to the cultures, and the effect on fsr gene expression was studied after an additional 30 min. Northern blot analyses show that glutamine strictly represses pigment biosynthesis, while the genes are still expressed if sodium nitrate or water is added (Fig. 5C).

Next, we wanted to determine whether single deletions of each of the fsr genes affect the expression of the other cluster genes, as has been shown for bikaverin genes (79). For this purpose, deletion mutants of all six genes were generated. Surprisingly, deletion of the PKS-encoding gene fsr1 resulted in complete downregulation of the other five fsr genes, while single deletions of the fsr2 to fsr5 genes did not affect the expression of any other pathway gene. The deletion of fsr6, encoding a putative Zn(II)2Cys6 transcription factor, resulted in the expected downregulation of all fsr genes, including fsr1 (see Fig. S6A in the supplemental material).

Chemical identification of intermediates and final products.

Cultivation of the wild type in ICI medium with sodium nitrate as the sole nitrogen source resulted in a complex spectrum of different pathway-specific compounds (Fig. 6A), all of which are missing in the Δfsr1 deletion mutant (Fig. 6B). The structure of the main compounds (except for compound 2, due to insufficient accumulation in the liquid culture) was extensively elucidated using their exact masses, characteristic UV spectra, and nuclear magnetic resonance (NMR) data. By these methods, we identified the previously described naphthoquinones 8-O-methylfusarubin (compound 1), 8-O-methylnectriafurone (compound 2), 8-O-methyl-13-hydroxynorjavanicin (compound 3), and 13-hydroxynorjavanicin (compound 5), along with one new compound, which was subsequently designated 8-O-methylanhydrofusarubinlactol (compound 4) (67, 7072). The structures identified were in agreement with published data. Additionally, we are presenting, for the first time, 13C NMR data for compounds 2 to 5 (for detailed spectroscopic data, see the supplemental material). These compounds and structurally related metabolites are known to be produced by different Fusarium spp. The first naphthoquinones with a similar structure were isolated and characterized by Arnstein et al. from Fusarium javanicum and were therefore designated javanicin and oxyjavanicin (2), the latter of which was later renamed fusarubin (63). O-Demethylanhydrofusarubin was the first and, until now, the only naphthoquinone pigment of the fusarubin family to be described in F. fujikuroi (21). To our knowledge, none of the other compounds presented here have been described in F. fujikuroi before.

Fig 6
Chemical analyses of fungal strains. For analyses of compounds, the indicated fungal strains were grown under conditions favoring fusarubin biosynthesis (ICI with 6 mM sodium nitrate). Samples were taken after 6 days of growth and were directly used for ...

To confirm that the six coregulated fsr genes are indeed involved in the biosynthesis or regulation of pigment production, the product spectra of the Δfsr1 to Δfsr6 mutants and of deletion mutants of the two border genes, FF_03983 and FF_03990 (Fig. 2B), were analyzed. The fact that the FF_03983 and FF_03990 deletion strains showed no alteration in the product spectra strengthened the suggestion made on the basis of our coregulation studies (Fig. 4) that these genes are not involved in the biosynthesis or regulation of the pigments (data not shown).

The pathway-related products synthesized by the Δfsr1 to Δfsr6 deletion mutants were analyzed in detail to demonstrate their involvement in the biosynthesis of fusarubins (Fig. 6B). The Δfsr1, Δfsr2, and Δfsr3 mutants are the only mutants showing an alteration in the product spectrum, indicating their role in the formation of the final products.

Under pigment-favoring conditions, three major peaks were accumulated by the Δfsr2 and Δfsr3 mutants; they are labeled as compounds 6 and 7 in the chromatogram of the Δfsr2 mutant and compound 8 in that of the Δfsr3 mutant. Compound 6 showed an NMR spectrum similar to that of 6-O-demethyl-5-deoxyfusarubin, previously identified from Nectria haematococca (55), but with slightly different but distinct chemical shifts resulting from the position of the hydroxyl group at C-10 instead of C-5. Hence, compound 6 was designated 6-O-demethyl-10-deoxyfusarubin. Compounds 7 and 8 gave similar NMR spectra of the polyketide backbone, without the hydroxyl groups at positions C-5 and C-10, where the A and B rings are already formed, but not the C ring. The structures show characteristic chemical shifts indicating the presence of an aldehyde, which was confirmed by 2-D NMR analyses. The only difference is the presence of an additional methyl group at position C-6 in compound 8. The two intermediates were therefore designated 6-O-demethylfusarubinaldehyde and fusarubinaldehyde, respectively (for detailed spectroscopic data, see the supplemental material). None of these three compounds have been described before.

In contrast, deletion of fsr4 or fsr5 did not alter the product spectrum of the metabolites, indicating that these genes are not involved in the modification of the PKS-derived precursor under these specific conditions. Interestingly, product formation was enriched in the Δfsr4 mutant compared to that in the wild type, suggesting that fsr4 might possess a regulatory function in the biosynthesis of fusarubins. Deletion of fsr6, a putative Zn(II)2Cys6 transcription factor, resulted in the total loss of the respective products (Fig. 6B), indicating its function as a pathway-specific transcription factor.

In order to identify the Fsr1-derived product, a mutant in which all genes except fsr1 and fsr6 are deleted (Δfsr2-5) was generated. Surprisingly, this deletion resulted not only in the downregulation of the deleted genes, fsr2 to fsr5, but also in that of the two remaining genes, fsr1 and fsr6 (see Fig. S6B in the supplemental material). To overcome this effect, an fsr1 overexpression vector (gpdprom:fsr1) was transformed into the Δfsr2-5 deletion mutant, resulting in Δfsr2-5/OE:fsr1 mutants overexpressing fsr1 under the control of the strong A. nidulans gpd (glucose-6-phophatase dehydrogenase) promoter. By use of this approach, one major compound accumulated and was found to be identical with 6-O-demethylfusarubinaldehyde (compound 7), found in the Δfsr2 mutant (Fig. 6C; for detailed information, see the supplemental material). This heptaketide, lacking the methyl group as well as the two hydroxyl groups at C-5 and C-10, is therefore the earliest intermediate in the biosynthetic pathway of the fusarubins.

The fusarubins are responsible for the pigmentation of the perithecia.

Due to the close relatedness of Fsr1 to the Pgl1 proteins of F. graminearum and F. verticillioides (28, 60) (Fig. 2A), we wanted to investigate whether the fusarubin-type compounds are identical with the yet unknown perithecial pigments in F. fujikuroi. Therefore, sexual crosses were performed between the wild-type strain IMI58289 (Mat-1) and F. fujikuroi strain C1995, with the opposite mating type (Mat-2), on the one hand, and between the IMI58289/Δfsr1 and C1995/Δfsr1 deletion mutants, on the other hand. For this purpose, the fsr1 knockout fragment was also transformed into C1995, and deletion mutants were identified by diagnostic PCR (data not shown). Microscopic analysis of the fruiting bodies resulting from both sexual crosses clearly showed that the loss of fsr1 in both mating partners resulted in colorless perithecia that lack the normal purple pigmentation (Fig. 7).

Fig 7
The fusarubins are responsible for the coloration of the fruiting bodies in F. fujikuroi. Strains were crossed as described in Materials and Methods. Perithecia are indicated by black arrows. Size standards are shown in the lower left corners. (A) Crossing ...


Polyketides represent a highly diverse group of natural products within fungal secondary metabolites. They are generated by large multidomain enzymes, the PKSs (14). Among them are naphthoquinones, a class of polyketides formed by NR-PKSs. They are often deeply colored, and many of them show phytotoxic, insecticidal, antibacterial, and fungicidal activities (52). Due to their structural properties, they are also thought to be involved in protection against extreme environmental conditions, such as UV irradiation and desiccation (30).

Until now, only the red polyketides bikaverin and norbikaverin (40), as well as O-demethylanhydrofusarubin (21), were identified as naphthoquinone pigments of F. fujikuroi. In this study, we describe the identification of new secondary metabolites with naphthoquinoid structures, as well as the corresponding biosynthetic genes, organized in a gene cluster in the genome of F. fujikuroi. These metabolites are red pigmented like bikaverin, but their production is differently regulated by external factors such as pH and nitrogen sources. The new red pigments were identified as members of the fusarubin family (Fig. 6D). Although most of these structures have been described previously in different Fusarium spp. (52), their biological function and biosynthetic genes have remained mysterious until now.

Fusarubin-type naphthoquinones are responsible for the coloration of the fruiting bodies.

In this study, we show for the first time that the fusarubin-type naphthoquinones (Fig. 6D) are identical with the perithecial pigments (Fig. 7). Perithecia are the sexual fruiting bodies in which the progeny of the fungus, the ascospores, are formed. Here naphthoquinones might function as protectants, as suggested for their role in F. solani (30), against harmful environmental conditions, such as reactive oxygen species (ROS), UV irradiation, desiccation, and fungivorous insects. In A. fumigatus, the conidia of mutants defective in PKS-derived pigment biosynthesis are more susceptible to killing by macrophages than are pigmented wild-type conidia (35). Furthermore, studies of A. nidulans have shown that certain insects preferentially feed on fungal mutants exhibiting a restricted secondary metabolite profile, indicating that a fungus capable of the production of its full secondary metabolite spectrum has an enhanced survival rate (61). The colorless perithecia of fsr1 mutants were larger than the colored perithecia produced by the wild type (Fig. 7), suggesting that the red pigments are also important for the integrity of the perithecial cell wall. A similar correlation was found for A. fumigatus, where PKS-derived melanin is required for the correct assembly of the cell wall layers in the conidia (59). Interestingly, in F. fujikuroi, the fusarubin-type pigments are not restricted to the fruiting bodies but also occur in the culture broth when the organism is grown in liquid culture. This raises the question of whether these secondary metabolites have further functions besides protection of the perithecia. One possibility would be a functional redundancy between the red pigments, since fusarubins and bikaverin are not produced under the same culture conditions in F. fujikuroi. A similar situation was found for F. graminearum, where deletion of the aurofusarin-specific PKS gene aur1 affected the expression of pgl1, which is responsible for the formation of pigmented perithecia (28).

Biosynthetic pathway of the fusarubins.

We identified the main compounds produced by the wild type as the naphthoquinones 8-O-methylfusarubin (compound 1), 8-O-methylnectriafurone (compound 2), 8-O-methyl-13-hydroxynorjavanicin (compound 3), 8-O-methylanhydrofusarubinlactol (compound 4), and 13-hydroxynorjavanicin (compound 5) (Fig. 6D) on the basis of their exact masses, characteristic UV fingerprints, and NMR data (67, 7072). All of these compounds, except for 8-O-methylanhydrofusarubinlactol (compound 4), which has never been described before, are known metabolites of other Fusarium spp. (52). However, this is the first report of the identification of these naphthoquinones in F. fujikuroi. The finding that these structurally related but distinctly different naphthoquinones are all products of the same NR-PKS might indicate that most of these structures identified in Fusarium spp. are intermediates or final products of one and the same biosynthetic pathway.

To analyze this pathway in detail, it is important to identify the first intermediate released by the responsible PKS. Fsr1 shows the typical domain organization of a nonreducing PKS, consisting of a SAT domain, a KS domain, a MAT domain, and two ACP domains, with one difference: the often-found TE or TE/CLC domain is replaced by an R domain (Fig. 2A). This finding led to the hypothesis that the PKS-derived heptaketide is most likely released as an aldehyde. By overexpressing fsr1 in the Δfsr2-5 mutant in an Fsr6-independent manner, we were able to identify the first intermediate of the pathway as 6-O-demethylfusarubinaldehyde (compound 7) (Fig. 6C and D). This reductive release mechanism, resulting in the formation of an aldehyde intermediate, has been shown previously for the 3-methylorcinaldehyde synthase Pks1 in Acremonium strictum (5) and AfoE, the NR-PKS of the asperfuranone pathway (13), which also harbor R domains. This mechanism has also been shown in bacteria, e.g., for Lgr, the nonribosomal peptide synthase (NRPS) of the gramicidin pathway in Bacillus brevus (38); for SmfC, one NRPS of the SFM-A gene cluster in Streptomyces lavendulae (45); and for MxcG, the NRPS responsible for the formation of the aldehyde intermediate in the myxochelin biosynthesis of Stigmatella aurantiaca (46). Furthermore, the α-aminoadipate reductase Lys2p, which also harbors an R domain, has been shown to be responsible for the formation of an aldehyde intermediate in the lysine-biosynthetic pathway of Saccharomyces cerevisiae (25). In the biosynthetic pathway of fusarubins, the A and B rings of compound 7 are most likely formed by a specific C-4/C-9-type aldol cyclization and aromatization catalyzed by the identified PT domain of Fsr1. This is in accordance with the mechanism that the PT domain of A. fumigatus PksA catalyzes during aflatoxin formation (15, 18, 47). Since phylogenetic analysis of the Fsr1 PT domain shows close relatedness to PksA (see Fig. S5 in the supplemental material), this C-4/C-9-type is very likely to occur in F. fujikuroi, but experimental proof is needed. Parisot et al. described a similar metabolite, 6-O-demethylnectriachrysone, isolated from F. solani mutants created by random UV mutagenesis (57). This slightly different structure most likely results from rearrangement of the released aldehyde to a more stable intermediate during the product isolation procedure, which was different from our method. This finding indicates that 6-O-demethylfusarubinaldehyde (compound 7), rather than the proposed fusarubinic acid (54), is also the first intermediate in F. solani, as was shown for F. fujikuroi in this paper. This is the first report of an NR-PKS harboring an R domain showing C-4/C-9 cyclization.

Although a SAT domain was identified in Fsr1, and compound 7 is most likely built by a C14 alkyl chain, it remains unclear whether this alkyl chain consists of malonyl-derived building blocks only or whether the SAT domain accepts a starter unit with a different chain length. Investigation of the SAT domain of A. fumigatus PksA provides experimental proof that this SAT domain accepts a hexanoyl starter unit (15). However, this hexanoyl starter is assembled by a fatty acid synthase (FAS) α-subunit (HexA) and a FAS β-subunit (HexB) that are encoded by genes in close proximity to pksA in A. fumigatus (78). Other examples of NR-PKSs harboring a SAT domain that are known to accept starter units other than malonyl-CoA are found in F. graminearum trichothecene and A. nidulans asperfuranone biosynthesis. In both cases, the NR-PKS accepts the product of a highly reducing PKS (HR-PKS) as a starter unit. Interestingly, the genes encoding the NR-PKS and the HR-PKS belong to the same gene cluster (13, 39), a situation resembling that of the aflatoxin gene cluster in A. fumigatus. In F. fujikuroi, no HR-PKS- or FAS-encoding genes are found in close proximity to the fusarubin gene cluster. However, the nature of the building blocks assembled by Fsr1 needs experimental proof. This would provide valuable information that might help answer the question whether the gene involved in starter unit formation needs to be located in the same gene cluster as the NR-PKS-encoding gene in case the starter unit is not malonyl-CoA.

The presence of a tandem ACP motif in Fsr1 was also observed in A. nidulans WA, where the two ACP domains were shown to be redundant (26), suggesting a similar situation for Fsr1 in F. fujikuroi.

To identify further intermediates of this pathway, single deletion mutants of all six pathway genes were generated, and the resulting metabolites were analyzed by HPLC-DAD (Fig. 6B) and HPLC-UV-FTMS (data not shown). Of all the mutants, only the Δfsr2 and Δfsr3 mutants showed a metabolite spectrum altered from that of the wild type. The structure of the accumulated intermediates was additionally elucidated by performing NMR experiments. By those experiments, 6-O-demethylfusarubinaldehyde (compound 7) and 6-O-demethyl-10-deoxyfusarubin (compound 6) were identified in the fsr2 deletion mutant, while fusarubinaldehyde (compound 8) was the main product of the fsr3 deletion mutant. These results confirmed the hypothesized functions of those two enzymes (Table 1) as an O-methyltransferase (Fsr2) and a monooxygenase (Fsr3), as previously proposed for mutants created by UV mutagenesis in F. solani (56, 57).

The intermediates found in the Δfsr2 and Δfsr3 mutants and putative intermediates described in the literature indicate that Fsr2 and Fsr3 function independently of each other. Nevertheless, the fact that the second hydroxyl group is not present at position C-10 in one of the Δfsr2 intermediates (compound 6) indicates that the methylation of the hydroxyl group at position C-6 is necessary for this step. Furthermore, methylation at C-8 takes place only if both hydroxyl groups at C-5 and C-10 are present and does not seem to be an essential biosynthetic step, as indicated by the accumulation of 13-hydroxynorjavanicin (compound 5) in the wild type. A similarly flexible pathway was shown for the biosynthesis of bikaverin, where the monomethylated form, norbikaverin, coexists with the dimethylated form, bikaverin, in liquid culture (79). The accumulation of only one intermediate in the Δfsr3 mutant, instead of the five structurally related but distinct metabolites found in the wild type, suggests that Fsr3 is responsible not only for the hydroxylation steps at positions C-5 and C-10 but also for the reduction of the released aldehyde as well as the oxidation and subsequent 13-hydroxylation after the cleavage of CO2. This series of possible biosynthetic steps indicates the role of Fsr3 as a multifunctional monooxygenase. So far, multiple oxidization reactions by a single enzyme have been described only for cytochrome P450 monooxygenases, including three of the four P450 monooxygenases in the gibberellin biosynthesis of F. fujikuroi (62, 76, 77) and Tri4 in the trichothecene biosynthesis of F. graminearum (73). In A. fumigatus, chain shortening of YWA1 was shown to be catalyzed by Ayg1, resulting in the formation of 1,3,6,8-tetrahydroxynaphthalene (T4HN) (27). Since no protein with significant homology to Ayg1 was shown to be encoded in the fsr gene cluster, a similar mechanism is very unlikely. However, whether Fsr3 is capable of multiple oxidation steps, including the cleavage of CO2, awaits experimental proof.

The proteins encoded by fsr4 and fsr5 show homologies to proteins harboring an oxidoreductase and a short-chain dehydrogenase domain, respectively. Surprisingly, no enzymatic function could be determined for either of the two, because deletion of their encoding genes did not alter the product spectrum (Fig. 6B). Remarkably, deletion of fsr4, but not fsr5, seems to enhance the biosynthesis of the products (Fig. 6B), although the fsr1 to fsr3 genes were not upregulated in the fsr4 deletion mutant at the time point investigated (see Fig. S6A in the supplemental material). Proteins harboring a short-chain dehydrogenase domain were previously shown to possess regulatory functions in filamentous fungi. Thus, the Nmr1 protein, which also has a predicted short-chain reductase domain, was linked to the nitrogen-dependent regulation of secondary metabolism in F. fujikuroi. Yeast two-hybrid analyses showed that Nmr1 interacts with the global nitrogen regulator AreA, thereby inactivating AreA under nitrogen-sufficient conditions, leading to a subsequent downregulation of genes responsible for gibberellin biosynthesis (65). Additionally, an nmr-like gene, bik4, was identified in the bikaverin gene cluster, and the encoded protein, Bik4, was suggested to be a coregulator of the Zn(II)2Cys6 transcription factor Bik5 (79). However, since fusarubin derivatives are produced in the perithecia under natural conditions, the possibility that Fsr4 and Fsr5 catalyze specific enzymatic steps during perithecium development cannot be ruled out.

Organization of the perithecial pigment gene cluster in F. fujikuroi and related species.

The gene cluster for the perithecial pigment in F. solani differs from the gene clusters found in F. verticillioides, F. graminearum (60), and F. fujikuroi (this paper). Remarkably, a core gene cluster consisting of genes encoding the PKS (Pgl1, Fsr1), the methyltransferase (Omt1, Fsr2), and the monooxygenase (Fdm1, Fsr3) is syntenic in all four fusaria, whereas the entire genomic region from the FF_03983 to the FF_03990 gene, including the cluster genes fsr1 to fsr6, is syntenic in F. verticillioides only (60). In F. graminearum and F. solani, the synteny between fsr3 and fsr4 is abrogated, suggesting that in these species, the respective perithecial pigment gene cluster may consist of genes different from those in F. fujikuroi. This constitutional difference of the respective gene cluster in F. solani and F. fujikuroi could account for the different production conditions and the slightly different metabolite spectrum for F. solani (described by Kurobane et al. [43]) versus F. fujikuroi (this study).

Interestingly, Ma et al., by using a microarray approach, described seven genes that were coregulated with pgl1 in F. verticillioides (49). However, in F. fujikuroi, only five of the homologous genes, fsr2 to fsr6, adjacent to the PKS-encoding fsr1 gene were found to be strictly coregulated (Fig. 4), possibly due to the different culture conditions used. The restriction of the fusarubin gene cluster in F. fujikuroi to the genes fsr1 to fsr6 is confirmed by the fact that deletion of the genes bordering fsr1 and fsr6 had no impact on biosynthesis, and these genes were not expressed under any of the conditions tested in this study (Fig. 4).

The two groups of red pigments in F. fujikuroi show contrasting regulation.

Interestingly, the two groups of pigments in F. fujikuroi, bikaverins and fusarubins, are found to be expressed under contrasting pH conditions. The most remarkable difference is that bikaverin is produced only under acidic conditions, while the fusarubin pigments are formed only when the pH of the culture medium is neutral or alkaline (Fig. 1). Due to the strict response of fsr gene expression to changing pH conditions, the involvement of the pH regulator PacC is currently under investigation. In A. nidulans, this C2H2 zinc finger transcription factor is known to preferentially activate target genes at an alkaline pH and to repress genes that are expressed at an acidic pH (58). For the bikaverin genes, noncanonical pH regulation has been demonstrated: the genes are repressed under alkaline conditions in a PacC-dependent manner (79).

Another interesting yet unknown mechanism for regulating the expression of bikaverin genes is their strict interdependency. If any of the biosynthesis genes bik1 to bik3 is deleted, the remaining genes also are not expressed, due to a yet unknown mechanism (79). This is not the case for the fsr genes, where only the deletion of the PKS-encoding gene, fsr1, or the gene coding for a Zn(II)2Cys6 transcription factor, fsr6, results in a total loss of gene expression; deletion of any other gene does not affect the expression of the remaining fsr genes (see Fig. S6A in the supplemental material). In addition to these contrasting regulation patterns, there is also a common regulation mechanism: large amounts of nitrogen repress the expression of bikaverin (79) as well as fusarubin (described here) biosynthetic genes. We clearly demonstrated that this repression is specifically triggered by glutamine (Fig. 5C). How glutamine sensing and subsequent signal transduction work in F. fujikuroi is under investigation.

In conclusion, we describe the identification of five new red pigments in F. fujikuroi that are produced under specific culture conditions (alkaline pH; nitrogen limitation) different from those for bikaverins. By a combination of HPLC, MS, and NMR analyses, we identified 8-O-methylfusarubin (compound 1), 8-O-methylnectriafurone (compound 2), 8-O-methyl-13-hydroxynorjavanicin (compound 3), 8-O-methylanhydrofusarubinlactol (compound 4), and 13-hydroxynorjavanicin (compound 5) as true metabolites of the pigment pathway in F. fujikuroi (67, 7072). Based on the newly identified structures, we were able to establish a biosynthetic pathway for fusarubin-like metabolites: the heptaketide backbone of the polyketide results from the condensation of seven acetyl-CoA subunits and is subsequently released by the PKS Fsr1 as 6-O-demethylfusarubinaldehyde (compound 7). After several methylation and hydroxylation steps by Fsr2 and Fsr3, respectively, the final products compounds 1 to 5 accumulate in the liquid culture of wild-type F. fujikuroi, with 8-O-methylfusarubin (compound 1) as the main product (Fig. 8).

Fig 8
Biosynthetic pathway of fusarubins in F. fujikuroi. Shown is the verified route of naphthoquinone formation in F. fujikuroi based on the main compounds identified in the liquid culture. The condensation of seven acetyl-CoA units results in the formation ...

In addition, we pinpointed these pigments for the first time to a concrete gene cluster in the genus Fusarium and demonstrated that the fusarubins described here are the formerly unknown perithecial pigments.

Supplementary Material

Supplemental material:


This work and the research fellowship of Lena Studt were supported by funds of the Deutsche Forschungsgesellschaft (DFG), Graduiertenkolleg 1409 (GRK1409, Germany).

We thank Barbara Howlett of the School of Botany, University of Melbourne, for providing the pZPnat1 vector; J. F. Leslie, Kansas State University, for F. fujikuroi strain C1995; Dominik Wagner for construction of the modified pRS426 vector; Katharina W. von Bargen for help with the acquisition of NMR data; and Kathleen Plamper for excellent technical support.


Published ahead of print 6 April 2012

Supplemental material for this article may be found at http://aem.asm.org/.


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