Figure 1.
Cluster of apicidin F in Fusarium fujikuroi and apicidin in F. semitectum and their corresponding structures.
(A) The apicidin F gene cluster (APF1-APF12) is located at the terminal part of chromosome I, thus it has only one border gene (FFUJ_00014). The arrows indicate the direction of transcription. In comparison to F. semitectum, F. fujikuroi is missing aps10 and aps2/APF2 & aps3/APF3 are orientated in an opposite manner. (B) Apicidin is produced by F. semitectum [14]. Apicidin F is produced by F. fujikuroi [10].
Table 1.
Gene name, accession numbers, length and predicted function of the apicidin F cluster and its border gene.
Figure 2.
Co-regulation of the apicidin F cluster genes.
The WT, ΔAPF2 (TF) and OE::APF2 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 APF genes 1–12 and the border gene FFUJ_00014 were used.
Figure 3.
Apicidin F cluster genes are expressed under high amounts of glutamine (gln) from the second to the third day.
(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. APF1 and APF9 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. APF6 and APF9 were used as probes.
Figure 4.
Regulation of the apicidin F cluster.
(A) The pH regulator PacC seems to be an activator of the apicidin F genes. The WT and ΔPACC 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, ΔAREA and ΔAREB and the gln auxotroph mutant ΔGLN1 were grown for three days in 60 mM gln. (C) The WT, ΔVEL1, ΔVEL2 and ΔLAE1 were grown for three days in 60 mM gln. APF6 and APF9 were used as probes for all Northern blot analyses.
Figure 5.
Over-expression of the transcription factor-encoding gene APF2 (OE::APF2) is able to overcome the nitrogen regulation of apicidin F.
(A) HPLC-DAD measurement of the extracted mycelium of the WT and the OE::APF2 mutant after three days. Both strains were grown in four nitrogen conditions, 6 and 60 mM glutamine (gln) and 6 and 120 mM NaNO3. Apicidin F was measured at a wavelength of 280 nm. (B) Northern blot analyses of the WT and the OE::APF2 mutant. Same conditions were used as for the HPLC measurements. APF2 and APF9 were taken as probes. (C) HPLC-DAD measurement of the extracted mycelium of the WT and the OE::APF2 mutant after seven days in 6 and 120 mM NaNO3. Product formation was assessed in triplicates and normalized to the WT level.
Figure 6.
The transcription factor (TF) Apf2 contains a basic DNA binding domain, four ankyrin repeats and is localized in the nucleus.
(A) ClustalW alignment with amino acids of Cochliobolus carbonum ToxE (AFO38874), Fusarium semitectum Aps2 (GQ331953) and Fusarium fujikuroi 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 [55]. (B) The TF was fused to green fluorescent protein (GFP) at the C-terminus. The ΔAPF2 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 File S1.
Figure 7.
Mutation of the putative “Api-box” motif in the promoter region of APF1 (NRPS) and APF11 (transporter) resulted in reduced production of apicidin F.
(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 APF1/APF11 promoter (P-mut1 and P-mut2, for the strategy see Fig. S3 in File S1). (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.
Figure 8.
Influence of single APF gene deletions on the expression of the remaining genes and the production of apicidin F in these mutants.
(A) The single gene deletions have no impact on the expression of the remaining genes. Only deletion of APF2 (TF) resulted in down-regulation of all genes except of APF3. For this experiment, the WT and the single deletion mutants of the apicidin F gene cluster were grown for three days in 60 mM glutamine (gln). After harvesting, a northern blot was performed and hybridized with indicated probes APF1, APF2, APF3, APF6, APF9 and APF11. (B) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of the APF gene cluster grown in ICI with 60 mM gln for three days. Shown are the extracted ion chromatograms for the [M+H]+-ion of apicidin F (646.3235±0.0032), the axes are normalized to the WT-level. The deletion mutant of the transporter-encoding gene (ΔAPF11) still produces WT-levels of apicidin F. ΔAPF3 produces apicidin F as well but in a decreased manner. Analysis of the mycelium extracts led to comparable results (Fig. S10 in File S1).
Figure 9.
Deletion of the cluster genes APF3 and APF9 revealed new analogs of the apicidin F biosynthetic pathway.
(A) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of APF3 (ΔAPF3) and APF9 (ΔAPF9) 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 ΔAPF9-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.
Figure 10.
Cytotoxicity of apicidin F and apicidin.
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).
Figure 11.
Proposed biosynthetic pathway of apicidin F.
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 [14]. In our studies, we could demonstrate that deletion of the P5C reductase-encoding gene APF3 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 N-methoxy-l-tryptophan. We suggest that one of the P450 oxidases (Apf7 or Apf8) N-oxidizes l-tryptophan and then the O-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 APF5 is involved in the synthesis of the octanoic acid backbone by fixing one acetyl-CoA unit and three malonyl-CoA units [14]. 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 F. semitectum, it could be shown that deletion of aps7 led to the production of apicidin E (lacks the keto group in comparison to apicidin) [14]. 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).