Regulation of the apicidin F cluster.

<p>(A) The pH regulator PacC seems to be an activator of the apicidin F genes. The WT and Δ<i>PACC</i> were grown for three days under optimal conditions (60 mM glutamine, gln). The cultures were harvested and after washing, the mycelium was shifted into new flasks containing 60 mM gln adjusted to an ambient pH of 4 or 8, respectively. After 2 h the cultures were harvested again. (B) The nitrogen regulators AreB and glutamine synthetase (GS) are activators of the apicidin F gene expression. The WT, Δ<i>AREA</i> and Δ<i>AREB</i> and the gln auxotroph mutant Δ<i>GLN1</i> were grown for three days in 60 mM gln. (C) The WT, Δ<i>VEL1</i>, Δ<i>VEL2</i> and Δ<i>LAE1</i> were grown for three days in 60 mM gln. <i>APF6</i> and <i>APF9</i> were used as probes for all Northern blot analyses.</p

Deletion of the cluster genes <i>APF3</i> and <i>APF9</i> revealed new analogs of the apicidin F biosynthetic pathway.

<p>(A) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of <i>APF3</i> (Δ<i>APF3</i>) and <i>APF9</i> (Δ<i>APF9</i>) grown in ICI with 60 mM glutamine for three days. Shown are the extracted ion chromatograms for the [M+H]⁺-ion of proline apicidin F (apicidin J, 632.3079±0.0032, left) and for the [M+H]⁺-ion the Δ<i>APF9</i>-product (apicidin K, 632.3443±0.0032, right). The axes are normalized to the WT-level. (B) Structures of the two identified products: apicidin J and apicidin K.</p

The transcription factor (TF) Apf2 contains a basic DNA binding domain, four ankyrin repeats and is localized in the nucleus.

<p>(A) ClustalW alignment with amino acids of <i>Cochliobolus carbonum</i> ToxE (AFO38874), <i>Fusarium semitectum</i> Aps2 (GQ331953) and <i>Fusarium fujikuroi</i> Apf2 (FFUJ_00012). Identical amino acids are highlighted in grey, the positions of the domains are highlighted in either orange (basic DNA binding domain) or green (four ankyrin repeats) and based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Pedley1" target="_blank">[55]</a>. (B) The TF was fused to green fluorescent protein (GFP) at the C-terminus. The Δ<i>APF2</i> mutant was used as background. The two strains were grown for one day in 60 mM glutamine. Size of scale bars is indicated. A supplemental figure with controls is depicted in Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>.</p

Co-regulation of the apicidin F cluster genes.

<p>The WT, Δ<i>APF2</i> (TF) and OE::<i>APF2</i> were grown for three days in 60 mM glutamine. RNA was isolated from lyophilized mycelia. Northern blot analysis was done as described in methods. As probes, the <i>APF</i> genes <i>1</i>–<i>12</i> and the border gene <i>FFUJ_00014</i> were used.</p

Mutation of the putative “Api-box” motif in the promoter region of <i>APF1</i> (NRPS) and <i>APF11</i> (transporter) resulted in reduced production of apicidin F.

<p>(A) Bioinformatic searches revealed an eight-base-pair motif with the consensus sequence 5′-TGACGTGA-3′ that was found in all promoters of the apicidin F cluster except in the promoter region of the transcription factor (TF)-encoding gene itself. In our study, we created two mutants with point mutations in the <i>APF1</i>/<i>APF11</i> promoter (P-mut1 and P-mut2, for the strategy see Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>). (B) Biosynthesis of apicidin F was monitored with HPLC-HRMS. After growth for three days in 60 mM glutamine, the cultures of the WT and the two mutants P-mut1 and P-mut2 were harvested. Apicidin F was extracted from lyophilized mycelium. 10 µL of a 1 µg/mL apicidin solution (internal standard) were added to 90 µL of the sample. For the calculation, the peak area of apicidin F [M+H]⁺ (646.3235±0.0032) was divided with that of apicidin [M+H]⁺ (624.3756±0.0032). Product formation was normalized to the WT level. Experiment was performed in a triplicate.</p

Cluster of apicidin F in <i>Fusarium fujikuroi</i> and apicidin in <i>F. semitectum</i> and their corresponding structures.

<p>(A) The apicidin F gene cluster (<i>APF1</i>-<i>APF12</i>) is located at the terminal part of chromosome I, thus it has only one border gene (<i>FFUJ_00014</i>). The arrows indicate the direction of transcription. In comparison to <i>F. semitectum</i>, <i>F. fujikuroi</i> is missing <i>aps10</i> and <i>aps2</i>/<i>APF2</i> & <i>aps3</i>/<i>APF3</i> are orientated in an opposite manner. (B) Apicidin is produced by <i>F. semitectum </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. Apicidin F is produced by <i>F. fujikuroi </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-vonBargen1" target="_blank">[10]</a>.</p

Apicidin F cluster genes are expressed under high amounts of glutamine (gln) from the second to the third day.

<p>(A) The WT was grown in four nitrogen conditions, 6 and 60 mM gln and 6 and 120 mM NaNO₃ for three days. After harvesting, RNA was isolated from the mycelium. <i>APF1</i> and <i>APF9</i> were used as probes. (B) The WT was grown from the second to the fifth day (d) in 60 mM glutamine. Northern blot analysis was performed with the extracted RNA. <i>APF6</i> and <i>APF9</i> were used as probes.</p

Proposed biosynthetic pathway of apicidin F.

<p>Based on our data, we postulated a biosynthetic pathway for apicidin F. Noteworthy, catalyzation steps which are marked with a “?” have not been confirmed experimentally. The NRPS as key enzyme incorporates four different amino acids to produce apicidin F. However, most of the amino acids are non-proteinogenic and therefore, have to be modified by other enzymes of the cluster. l-Phenylalanine is the only proteinogenic amino acid and directly used by Apf1. l-Pipecolic acid is the second precursor of Apf1 and is epimerized to D-pipecolic acid, probably by Apf1 activity, prior to incorporation. It is proposed that lysine is converted to Δ¹-pyrroline-5-carboxylate (P5C). A P5C reductase catalyzes the transformation to proline. This enzyme could also convert Δ¹-pyrroline-6-carboxylate (P6C) to l-pip <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. In our studies, we could demonstrate that deletion of the P5C reductase-encoding gene <i>APF3</i> led to the incorporation of proline instead of pip resulting in the production of apicidin J. We claim, therefore, that this enzyme is responsible for the conversion of P6C into l-pip. The NRPS itself has an epimerization domain for the epimerization of l-pip into d-pip. Furthermore, Apf1 incorporates <i>N</i>-methoxy-l-tryptophan. We suggest that one of the P450 oxidases (Apf7 or Apf8) <i>N</i>-oxidizes l-tryptophan and then the <i>O</i>-methyltransferase Apf6 is able to catalyze the methylation of the hydroxy group. The fourth amino acid that is incorporated by Apf1 is l-2-aminooctanedioic acid. It is predicted that the fatty acid synthase-encoding gene <i>APF5</i> is involved in the synthesis of the octanoic acid backbone by fixing one acetyl-CoA unit and three malonyl-CoA units <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. Then one of the P450 oxidases may oxidize this backbone to 2-oxooctanoic acid. The aminotransferase Apf4 is predicted to catalyze the exchange of the keto group with an amino group. The next step would be the oxidation of 2-aminooctanoic acid by one of the P450 oxidases (Apf7 or Apf8). For <i>F. semitectum</i>, it could be shown that deletion of <i>aps7</i> led to the production of apicidin E (lacks the keto group in comparison to apicidin) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. We suggest that the last step is the oxidation of 2-amino-8-hydroxyoctanoic acid to 2-aminooctanedioic acid by the FAD-dependent monooxygenase Apf9 because deletion of the corresponding gene led to the production of apicidin K (lacks the acid group and has a hydroxyl group instead).</p

Gene name, accession numbers, length and predicted function of the apicidin F cluster and its border gene.

<p>Gene name, accession numbers, length and predicted function of the apicidin F cluster and its border gene.</p

Cytotoxicity of apicidin F and apicidin.

<p>Hep G2 cells were incubated with concentrations from 0.001 µg/mL to 100 µg/mL apicidn F or apicidin, respectively for 48 h. Cytotoxicity was determined using the CCK-8 assay. The values of the samples are shown in comparison to the solvent treated negative control (100%). Values are means ± S.D. (n = 9 samples). The letters a-d indicate four groups of samples which differ significantly from the samples labeled with a different letter according to the ANOVA with the Tukey post hoc test (p≤0.05).</p