Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides
© The Author(s) 2018
Received: 30 November 2017
Accepted: 3 February 2018
Published: 2 March 2018
Fungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus Aspergillus niger was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in A. niger, and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives.
The beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350–600 mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective A. niger expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products.
In this study, we show that the product portfolio of A. niger can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of A. niger to produce a range of different peptide natural products in titres high enough to become industrially relevant.
At module 1, a d-hydroxycarboxylic acid is activated at the adenylation domain (A1) and covalently bound to the peptidyl carrier protein domain (PCP1). The l-amino acid is activated at the A-domain of module 2 (A2), which is bound to the adjacent PCP2a domain and methylated at the methylation domain (Mt). For substrate elongation, two different models have been proposed. At the “parallel” model, the depsipeptide chain grows by the addition of a dipeptidol, consisting of a hydroxy acid and an N-methyl amino acid, previously coupled in the C2 domain. In this model, either PCP2a or PCP2b act as a so-called waiting position until the next dipeptidol is formed. Ester bond formation as well as macrocyclization occurs at the C3 domain. However, based on recent results by Yu et al.  and from our group , experimental evidence points to the “linear” or “looping” model: the elongation occurs by the attachment of a single building block (hydroxy acid or N-methyl amino acid), while the growing depsipeptide chain is passed between the PCP1 and the PCP2a/b domains. Peptide bond formation is catalysed in the C2 domain, while the C3 domain catalyses ester bond formation and macrocyclization. In this model, the role of the double PCP2a/b domains remains unclear, as either one of the domains is sufficient for biosynthesis of the final product [11, 12]. It was proposed that C1 has no direct catalytic function, because truncated CDP synthetases missing the C1 domain are still functional. Thus, C1 could rather have a stabilizing or supportive role during the catalytic cycle . Currently, it is not clear which of these two models accurately represents NRPs activity, and more investigations are required to fully understand the underlying mechanism of CDP biosynthesis. Due to their high degree of similarity, fungal CDP synthetases are ideal systems for combinatorial biosynthesis approaches, such as module and domain swapping, to obtain novel ‘new-to-nature’ compounds [11–14].
One option for obtaining fungal CDPs is via chemical synthesis, and several such strategies have thus far been described. However, N-methylation of amino acids, racemisation during the coupling of hydroxyl acids, as well as the final cyclization step, severely limit the effectiveness of these approaches . For instance, an improved protocol for the total synthesis of enniatin established by Ley and co-workers requires nine steps and results in an overall yield of 15% . Recently, the same group established a protocol based on flow chemistry and were able to synthesize different natural and unnatural CDPs with higher yields (32–52%) . However, high amounts of solvents and costly catalysts make this process uneconomical. A novel chemical synthesis approach using salt additives to support ring formation has been described to synthesize bassianolide, its closely related CDP verticilide , and a number of unnatural CDPs with varying ring sizes . Although yields of bassianolide (9%) were almost twice as high as for the first total synthesis published (5.9%) , these overall yields are comparably low for production purposes. An alternative and more sustainable way to produce CDPs (and more generally natural products) is by using a biotechnological approach. Here, it is advantageous to transfer the biosynthetic pathway of the natural product of interest from a microbiologically challenging, genetically intractable, or even pathogenic organism into a safe, genetically amendable and industrially established heterologous production host. In the case of CDPs, natural production strains have been established and the highest titres reported for beauvericin production by Fusarium oxysporum KFCC 11353P and Fusarium redolens Dzf2, which range between 400 and 420 mg/L, respectively [20, 21]. However, not many tools for their genetic modification are available. Production of fungal CDPs in heterologous bacterial hosts has been established, but only low titres were achieved. In the case of beauvericin biosynthesis in Escherichia coli, only 8 mg/L were produced . Additionally, enniatin production using Bacillus subtilis yielded titres which were also only in the mg/L range . Encouragingly, when Saccharomyces cerevisiae was used as heterologous host, higher CDP titres were reported: 74.1 mg/L for beauvericin and 26.7 mg/L for bassianolide . Recently, we were able to show that the industrial fungus Aspergillus niger, well-known for its high level production of organic acids and secreted proteins , is a promising host for heterologous production of enniatin. In this study, the ESyn encoding gene was put under control of the inducible Tet-on expression system  allowing high enniatin titres up to 4.5 g/L upon addition of the inducer doxycycline (Dox) . This strain relies on feeding with the substrate d-hydroxy isovalerate, as it lacks the ketoisovalerate reductase gene kivR responsible for the generation of d-Hiv from 2-ketoisovalerate . Autonomous expression strains of A. niger independent of d-Hiv feeding were additionally established. In these strains, the kivR gene was either monocistronically or polycistronically co-expressed with the ESyn gene [27, 29].
In the present study, we determined whether the Beauveria bassiana CDPs beauvericin and bassianolide can also be produced in A. niger with high titres. Furthermore, we aimed to test whether the A. niger production strains, which lack the ketoisovalerate reductase gene kivR, can be used to generate new-to-nature beauvericin derivatives by precursor directed biosynthesis, which ultimately generated CDP variants that are accessible for further downstream chemical modifications.
Results and discussion
Generation of A. niger strains expressing BeauvSyn and BassSyn
Aspergillus niger is an excellent production organism for the synthesis of the hexamer enniatin, which consists of the two building blocks l-valine (l-Val) and d-hydroxy isovalerate (d-Hiv). To show that other CDPs relying on different precursor compositions or different ring sizes can be produced with high titres, our aim was to establish new production strains in an analogous fashion. Therefore, the Tet-on driven expression plasmids pDS8.2 (harbouring bbBeas encoding BeauvSyn), and pSB22.3 (harbouring bbBsls encoding BassSyn), were constructed and transformed into the A. niger strain AB1.13 (see Methods). This isolate is a useful production platform due to reduced protease activities . Transformants carrying a single copy of the expression constructs integrated at the pyrG locus were verified by PCR and Southern blot (Additional file 1: Figure S1). Positive strains were cultivated as previously described , specifically in 20 mL media in shake flasks, which were then tested for production of the respective CDP. The metabolites were extracted from the dried biomass of the transformants and analysed by LC–MS. The identity of beauvericin and bassianolide was verified by tandem mass spectrometry (Additional file 1: Figures S2 and S3). The relative amounts of produced beauvericin and bassianolide were quantified by multiple reaction monitoring mass spectrometry and the beauvericin-producing strains DSc1.4 (single integration) and DSc1.5 (tandem integration), as well as the bassianolide-producing strain SB19.23 (single integration), which were each selected for further analysis.
Medium optimization and CDP purification
Titres of beauvericin and bassianolide obtained in shake flask cultivations of A. niger
Concentration of amino acid and hydroxy acid precursor (mM)
0.45 ± 0.13 (DSc1.4)
1.04 ± 0.33 (SB19.23)
83.42 ± 5.24 (DSc1.4)
45.90 ± 11.05 (SB19.23)
293.62 ± 186.46 (DSc1.4)
378.77 ± 59.74 (SB19.23)
628.4 ± 211.1 (DSc1.5)
For purification of beauvericin and bassianolide, each 5 × 200 mL shake flask cultivations of strains DSc1.5 and SB19.23 were performed. Gene expression and CDP biosynthesis was induced 16 h post inoculation by the addition of 20 µg/mL Dox and each 15 mM of the respective precursors. Biomass was harvested after an overall cultivation time of 96 h, and beauvericin and bassianolide purified as described in the Methods section. The fractions of the HPLC runs containing only the respective CDP (Additional file 1: Figure S4), were pooled, acetonitrile evaporated, and the residues freeze dried. Overall, 306 mg of beauvericin and 172 mg of bassianolide could be purified from each 1 L culture medium. 1H-NMR spectra were recorded for both compounds and verified their purity. The signals obtained (Additional file 1: Figure S7) are in full accordance with data from the literature [7, 34].
Bioreactor scale production of CDPs
Bioreactor cultivations allow tight control of culture conditions (e.g. temperature, pH, dissolved oxygen), and a better nutrient uptake compared to shake flask cultivations, and are thus better suited to perform highly reproducible fermentations. We thus carried out eight independent bioreactor runs in order to analyse the performance and productivity of the single copy beauvericin-producing strain DSc1.4 and the single copy bassianolide-producing strain SB19.23 in more detail.
Titres of beauvericin and bassianolide obtained from bioreactor cultivations
Maximum titre (mg/L)
Beauvericin (run 1/run 2)
Bassianolide (run 1/run 2)
Standard cultivation condition
Precursor addition after 0 h
No pH control
Generation of non-natural CDP derivatives
We next tested whether the beauvericin and bassianolide producing A. niger strains DSc1.4 and SB19.23 can be used to produce new-to-nature beauvericin and bassianolide derivatives by exploiting the relaxed substrate specificity of A1 domains towards d-Hiv [44–46]. In non-autonomous production strains, substrate analogues do not compete with the natural substrates, and a precursor directed biosynthesis approach can therefore be applied [47, 48]. This technique, also called mutasynthesis or mutational biosynthesis, has been previously applied to obtain non-natural beauvericins from the heterologous host E. coli, as well as from a kivR deletion mutant of the natural producer B. bassiana. However, the titres for most of the non-natural beauvericin analogues stayed in the low mg/L range and only beauvericin analogues could be isolated when the alternative hydroxy acid displayed similar properties as d-Hiv (aliphatic side chains) [45, 47].
Because feeding of bromo-lactate resulted in significant amounts of bromo-beauvericin, we wanted to assess the capacity of this approach for production and isolation on a larger scale. As a preliminary experiment, different concentrations of the racemic precursor d/l-bromo-lactate (5, 10 and 15 mM) were added to DSc1.4 in small scale cultivations (20 mL scale). The addition of 10 mM of d/l-bromo-lactate gave higher titres than the addition of 5 mM, while no bromo-beauvericin could be detected in the cultures supplemented with 15 mM of the hydroxy acid. This coincided with significant less biomass formation, suggesting that high concentrations of bromo-lactate have toxic effects on A. niger. Based on these results, a concentration of 10 mM of d/l-bromolactate was chosen for large scale cultivation in shake flasks. 12 mg of pure bromo-beauvericin (preparative HPLC) could be successfully obtained as a colourless powder from a 1.1 L of culture of DSc1.4, the purity of which was proven by mass spectrometry and NMR (Additional file 1: Figures S9 and S10). The purified bromo-beauvericin was tested for antimicrobial and antiparasitic activity together with purified enniatin B, beauvericin and bassianolide. While bromo-beauvericin did not show any improved antimicrobial or antiparasitic activity compared to the other compounds, it interestingly did not show any cytotoxic effects against a mammalian cell line at a concentration of 100 µg/mL, whereas the IC50 value of natural beauvericin is 1.52 µg/mL. It is thus worth studying bromo-beauvericin further as a potential future antiparasitic drug (Additional file 1: Table S4).
Generation of deuterated CDP standards
Fungal CDPs are not only of pharmaceutical interest as lead structures, but are also prominent contaminants (especially enniatins and beauvericin) of food and feed, as most of their natural producers are plant pathogenic fungi [50–52]. Thus, robust, fast, and exact analytical methods are needed to detect and quantify these compounds, even in trace amounts, in both food and feed products suspected to be spoiled by fungi. Most described protocols are based on LC–MS measurements in combination with an external standard calibration curve [53–56]. However, these methods are only exact to a certain degree as they do not consider effects of the matrix which can lead to ion suppression or ion enhancement [57, 58]. Furthermore, the recovery rates of the analytes from biological samples may vary, which would also lead to altered results . Stable isotope dilution assays are superior to methods using external standards as they guarantee exact quantifications also of mycotoxins in grain products [58–61]. The biosynthesis of 15N3-labelled standards of enniatins and beauvericin in F. sambucinum (enniatin producer) and F. fujikuroi (beauvericin producer) grown on Na15NO3 as sole nitrogen source has been reported . With 430 µg (enniatin A), 450 µg (enniatin A1), and 1460 µg (beauvericin) of 15N-labelled compound purified from 500 mL of culture, titres are low while the price of the medium is relatively high.
Because sample preparation for LC–ESI–MS analysis from complex matrices (e.g. fungal biomass or grain products) can be laborious and time-consuming, especially if many samples need to be tested, it was evaluated whether the deuterated beauvericin and bassianolide standards could also be used for quantification on a MALDI-TOF instrument. In contrast to LC–ESI–MS, where the samples have to be pre-purified in order to keep the ion source clean, crude extracts can be directly applied to MALDI-TOF. To test this, defined amounts of deuterated beauvericin and bassianolide were added to a dilution series of the unlabelled compounds (Additional file 1: Table S5). The ratio of the peak areas of the sodium adducts (most abundant peaks) of the respective labelled and unlabelled compounds were plotted against the ratio of the concentration of the compounds to determine if a linear relation was observed. The ratios of the concentrations and the peak areas indeed show a linear correlation (Additional file 1: Figure S11). However, as pointed out by the coefficients of determination (R2 = 0.946 for the beauvericin measurements and R2 = 0.988 for the bassianolide measurements), quantification of both compounds is not exact. One problem of the MALDI-TOF measurements is that only a direct MS is being recorded and that MS/MS of labelled and unlabelled compound cannot be concomitantly measured. Thus, any compound showing the same or a very similar m/z value to the labelled or unlabelled analytes would alter the results. Nevertheless, the MALDI-TOF measurement is an interesting alternative for high-throughput applications where an approximate estimation of CDP concentrations can be tolerated (e.g. for screening many samples or strains). For exact measurements however, LC–MS/MS remains the method of choice as reviewed in .
Summary of highest CDP titres ever reported for bacterial and fungal expression hosts
5 g/L (enniatin B and other derivatives)
Shake flask cultivation
Shake flask cultivation
Shake flask cultivation
Shake flask cultivation
74.1 ± 0.3 mg/L
Shake flask cultivation
628.4 ± 211.1 mg/L
Shake flask cultivation
22.3 ± 1.5 mg/L
Shake flask cultivation
Shake flask cultivation
18.2 ± 0.6 mg/L
In shake flask cultivation
26.7 ± 2.8 mg/L
Shake flask cultivation
378.77 ± 59.74 mg/L
Shake flask cultivation
Strains and general cloning procedures
Plasmids, primers and strains used in this study are summarized in Additional file 1: Tables S1–S3. Molecular techniques for E. coli followed protocols described earlier . A. niger transformation and genomic DNA extraction from selected transformants was done according to . The BeauvSyn and BassSyn encoding genes bbBeas (GenBank accession number EU886196) and bbBsls (GenBank accession number FJ439897) were amplified from the genomic DNA of B. bassiana ATCC 7159 using the primer pairs Beauv_InFusion1_fw/Beauv_InFusion3_rv for bbBeas and Bass_InFusion1_fw/Bass_InFusion3_rv for bbBsls, respectively. The amplicons were ligated into the cloning vector pJET2.1 (Thermo Fisher Scientific Inc.), resulting in pDS2.1 (harbouring bbBeas) and pDS1.9 (harbouring bbBsls) and verified by restriction analysis and sequencing. Direct cloning of the full-length genes into the A. niger Tet-on expression vector pVG2.2 was not successful. Thus, the genes were split into three parts of approximately 3 kbp length and 15 bp overhangs to each other and the PmeI-linearized vector pVG2.2. pDS2.1 and pSB1.9 were used as templates and primer pairs Beauv_InFusion1_fw/Beauv_InFusion1_rv, Beauv_InFusion2_fw/Beauv_InFusion2_rv, Beauv_InFusion3_fw/Beauv_In-Fusion3_rv for bbBeas and Bass_InFusion1_fw/Bass_InFusion1_rv, Bass_In-Fusion2_fw/Bass_InFusion2_rv, Bass_InFusion3_fw/Bass_InFusion3_rv for bbBsls to amplify the respective gene fragments. The amplicons were ligated and assembled into the PmeI-linearized Tet-on expression plasmid pVG2.2 via the In-Fusion® HD Cloning Kit (Clontech), resulting in plasmids pDS8.2 (harbouring bbBeas) and pSB22.3 (harbouring bbBsls).
Shake flask cultivations of A. niger
For production of CDPs, transformants were cultivated in 20 mL or 200 mL enniatin production medium (EM) as described in  if not indicated otherwise. Hydroxy and amino acid precursors were added in the range of 0–25 mM. Cultures were inoculated with 5 × 106 spores/mL and Tet-On driven expression induced with 20 µg/mL doxycycline 16 h after inoculation.
Bioreactor cultivations of A. niger
Submerged cultivations were performed with Biostat bioreactors (Sartorius, Göttingen, Germany, 4 L working volume) as described before . Glucose-limited batch cultivation was initiated by inoculation of fermentation medium (CM with 5% of glucose: 7 mM KCl, 11 mM KH2PO4, 70 mM NaNO3, 2 mM MgSO4, 1x trace element solution , 0.1% casamino acids, 0.5% yeast extract, 5% glucose) with conidial suspension of A. niger transformants to give 109 conidia L−1. Glucose was sterilized separately from the fermentation medium. Temperature of 26 °C and pH 3 were kept constant if not stated otherwise, the latter by computer controlled addition of 2 M NaOH or 1 M HCl. Computer-controlled base addition to the culture broth was used as an indirect growth measurement . When the culture reached the early exponential growth phase (about 16 h after inoculation, corresponds to 1 g biomass dry weight kg−1), Dox (20 μg/ml), d-Hiv (15 mM) and l-Phe or l-Leu (15 mM) were added.
Purification of CDPs
Purification of CDPs from A. niger biomass was adapted from . In brief, the mycelium from a 1 L culture was harvested by suction filtration and lyophilized. The dried mycelium was ground in a mortar and extracted three times with 300 mL of EtOAc. The solvent was evaporated and the brownish residue filtered over a short silica column (n-hexanes/EtOAc = 50:50). The solvents were evaporated and the residues resolved in methanol. Insoluble residues were removed by filtration and the solvent evaporated. For beauvericin and bassianolide purification, the residues were dissolved in acetonitrile/water (80:20) and the solution was centrifuged at 10,000×g for 15 min to remove insoluble particles. The supernatant was subjected to reversed phase chromatography using a GROM-Sil 120 ODS-5 HE (10 µm, 250 × 20 mm) column on an Agilent 1100 series preparative HPLC system running isocratically on acetonitrile (+ 0.1% formic acid)/water (+ 0.1% formic acid) (70:30) for beauvericin or with a linear gradient (70–100% acetonitrile over 15 min) for bassianolide with a flow rate of 15 mL/min. For bromo-beauvericin purification, the residues were resolved in MeOH and subjected to reversed phase chromatography using a GROM-Sil 120 ODS-5 HE (10 µm, 250 × 20 mm) column on an Agilent 1100 series preparative HPLC system running isocratically on MeOH (+ 0.1% formic acid)/water (+ 0.1% formic acid) (81.5/18.5) with a flow rate of 15 mL/min. Fractions containing the respective CDP were pooled, acetonitrile and MeOH were evaporated and water was removed by freeze drying.
Analysis and quantification of produced CDPs
Biomass (which included in the case of shake flask cultivations also insoluble parts, i.e. talc particles) of a defined amount of culture broth was harvested by suction filtration and lyophilized and weighed. The biomass was ground and 25 mg were transferred to a 2 mL test tube and extracted with 1 mL of EtOAc, shaking overnight. The tubes were centrifuged at 13,000×g and 700 µL of the extract were transferred to a new 1.5 mL test tube and evaporated. The residues were dissolved in 1 mL of water/isopropanol (50:50), diluted if necessary and the amount of CDPs quantified in MRM mode on an ESI-Triple-Quadrupol-MS 6460 Series (Agilent Technologies) coupled to an Agilent 1290 Infinity HPLC system (Agilent Technologies) equipped with an Agilent Poroshell 120 EC-C18 (3.0 × 50 mm) column (Agilent Technologies), heated to 50 °C. The mobile phases were H2O (A) and isopropanol (B). The injection volume was set to 3 µl and the flow rate was 0.4 ml/min. The applied gradient was: 50–100% (0.0–3.2 min), 100% (3.2–4.5 min), 100–5% (4.5–4.6 min), 5% (4.6–5.6 min), 5–50% (5.6–5.7 min), 50% B (5.7–7.0 min). For beauvericin quantification, the m/z value for the precursor ion was set to 806.4 ([M + Na]+ adduct) and for the fragment ion to 384.1 as quantifier, for bassianolide quantification, the m/z value for the precursor ion was set to 931.6 ([M + Na]+ adduct) and for the fragment ion to 350.1 as quantifier. For every set of measurements, a new calibration curve was made using beauvericin or bassianolide isolated from A. niger transformants as an external standard. Peak areas were determined by manual integration using MassHunter Workstation Qualitative Analysis (Agilent Technologies). Exact masses of purified CDPs were recorded on an ESI-LTQ-Orbitrap-MS, Orbitrap XL (Thermo Fisher Scientific). Samples were dissolved in MeOH and measured by direct injection. Analysis was performed with the Xcalibur 2.2 software (Thermo Fisher Scientific). Retention times of labelled and unlabelled beauvericin and bassianolide were determined on an ESI-Orbitrap-MS, Exactive (Thermo Fisher Scientific) coupled to an Agilent 1260 Infinity HPLC system (Agilent Technologies) equipped with an Agilent Poroshell 120 EC-C18 (2.1 × 50 mm) column (Agilent Technologies). The mobile phases were H2O + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The injection volume was set to 2 µl and the flow rate was 0.4 ml/min. The applied gradient was: 40% (0.0–0.5 min), 40–100% (0.5–12.0 min), 100% (12.0–13.5 min), 100–40% (13.5–13.6 min), 40% B (13.6–15.5 min). 1:1 mixtures of labelled and unlabelled beauvericin and bassianolide were injected and retention times of the sodium adducts of each compound determined. Analysis was performed with the Xcalibur 2.2 software (Thermo Fisher Scientific).
For MALDI-TOF analysis, 1 µL of purified CDPs or crude extracts of A. niger transformants, solved in MeOH or acetonitrile, were either mixed with 1 µL of saturated 2,5-dihydroxybenzoic acid (DHB) or α-cyano-4-hydroxycinnamic acid (CHCA) solution [dissolved in an acetonitrile–water mixture (1:1), acidified with formic acid (1%)]. 1 µL of the mixture was spotted onto a ground steel MALDI target plate and allowed to dry and crystallize. Measurements were carried out on a Bruker ultrafleXtreme MALDI-TOF–MS, equipped with a smartbeam II laser. The intensity of the laser was set to 50% with a frequency of 2 kHz. Calibration was done with the peptide calibration standard (Bruker). Analysis was performed with the Compass for flexSeries 1.4 software (Bruker).
For NMR analysis, purified CDPs were solved in CDCl3 or MeOH-d4 and 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance III 700 MHz NMR spectrometer or Bruker Avance II 400 MHz NMR spectrometer. The signals of the non-deuterated solvent rests were used as standards. Chemical shifts are given in δ-units (ppm) relative to the solvent signal.
Synthesis of α-hydroxy acid precursors
Quantification of nitrate and l-Phe
Quantification of nitrate and l-Phe concentrations in the cultivation medium was performed with the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical) and the Phenylalanine Assay Kit (Sigma-Aldrich) according to the manufacturers’ protocols.
Antimicrobial and antiparasitic test assays
Bioactivity assays were performed as described in .
SB, VM and RDS designed the experiments, SB and DS conducted the plasmid construction and generation of the A. niger transformants, SB purified beauvericin and bassianolide and deuterated variants thereof from A. niger and performed the quantification and analysis of generated CDPs, SB and TS performed the bioreactor runs, SB, DP, TS and LR designed and analysed the MALDI-TOF quantification, SG and LA synthesized d-Hiv and hydroxy acid analogues, SB and LA performed the generation and purification of beauvericin derivatives, DK performed the synthesis of d/l-Hiv-d6. All authors interpreted and discussed the results. SB, VM and RDS prepared the manuscript. All authors read and approved the final manuscript.
We thank Dr. Guido Schiffer from Bayer Animal Health and Dr. Marcel Kaiser from the Swiss Tropical and Public Health Institute Basel for performing antimicrobial and antiparasitic test assays. We thank Dr. Andi Mainz for helping with the NMR analyses.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This work was supported by the Cluster of Excellence “Unifying Concepts of Catalysis” (UniCat) granted by the German Research Council (DFG) and the Marie Curie Initial Training Network QuantFung (607332) supported by FP7-PEOPLE-2013-ITN.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Süssmuth R, Müller J, von Döhren H, Molnár I. Fungal cyclooligomer depsipeptides: from classical biochemistry to combinatorial biosynthesis. Nat Prod Rep. 2011;28:99–124. https://doi.org/10.1039/c001463j.View ArticlePubMedGoogle Scholar
- Sivanathan S, Scherkenbeck J. Cyclodepsipeptides: a rich source of biologically active compounds for drug research. Molecules. 2014;19:12368–420. https://doi.org/10.3390/molecules190812368.View ArticlePubMedGoogle Scholar
- Sy-Cordero AA, Pearce CJ, Oberlies NH. Revisiting the enniatins: a review of their isolation, biosynthesis, structure determination and biological activities. J Antibiot (Tokyo). 2012;65:541–9. https://doi.org/10.1038/ja.2012.71.View ArticleGoogle Scholar
- Shekhar-Guturja T, Gunaherath GMKB, Wijeratne EMK, et al. Dual action antifungal small molecule modulates multidrug efflux and TOR signaling. Nat Chem Biol. 2016;12:867–75. https://doi.org/10.1038/nchembio.2165.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen B-F, Tsai M-C, Jow G-M. Induction of calcium influx from extracellular fluid by beauvericin in human leukemia cells. Biochem Biophys Res Commun. 2006;340:134–9. https://doi.org/10.1016/j.bbrc.2005.11.166.View ArticlePubMedGoogle Scholar
- Jow G-M, Chou C-J, Chen B-F, Tsai J-H. Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium. Cancer Lett. 2004;216:165–73. https://doi.org/10.1016/j.canlet.2004.06.005.View ArticlePubMedGoogle Scholar
- Zhan J, Burns AM, Liu MX, et al. Search for cell motility and angiogenesis inhibitors with potential anticancer activity: beauvericin and other constituents of two endophytic strains of Fusarium oxysporum. J Nat Prod. 2007;70:227–32. https://doi.org/10.1021/np060394t.View ArticlePubMedPubMed CentralGoogle Scholar
- Dornetshuber-Fleiss R, Heilos D, Mohr T, et al. The naturally born fusariotoxin enniatin B and sorafenib exert synergistic activity against cervical cancer in vitro and in vivo. Biochem Pharmacol. 2015;93:318–31. https://doi.org/10.1016/j.bcp.2014.12.013.View ArticlePubMedGoogle Scholar
- Heilos D, Rodríguez-Carrasco Y, Englinger B, et al. The natural fungal metabolite beauvericin exerts anticancer activity in vivo: a pre-clinical pilot study. Toxins (Basel). 2017;9:258. https://doi.org/10.3390/toxins9090258.View ArticleGoogle Scholar
- Süssmuth RD, Mainz A. Nonribosomal peptide synthesis—principles and prospects. Angew Chem Int Ed Engl. 2017;56:3770–821. https://doi.org/10.1002/anie.201609079.View ArticlePubMedGoogle Scholar
- Yu D, Xu F, Zhang S, Zhan J. Decoding and reprogramming fungal iterative nonribosomal peptide synthetases. Nat Commun. 2017;8:15349. https://doi.org/10.1038/ncomms15349.View ArticlePubMedPubMed CentralGoogle Scholar
- Steiniger C, Hoffmann S, Mainz A, et al. Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering. Chem Sci. 2017;8:7834–43. https://doi.org/10.1039/C7SC03093B.View ArticlePubMedPubMed CentralGoogle Scholar
- Yu D, Xu F, Gage D, Zhan J. Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases. Chem Commun (Camb). 2013;49:6176–8. https://doi.org/10.1039/c3cc42425a.View ArticleGoogle Scholar
- Zobel S, Boecker S, Kulke D, et al. Reprogramming the biosynthesis of cyclodepsipeptide synthetases to obtain new enniatins and beauvericins. ChemBioChem. 2016;17:283–7. https://doi.org/10.1002/cbic.201500649.View ArticlePubMedGoogle Scholar
- Hu DX, Bielitza M, Koos P, Ley SV. A total synthesis of the ammonium ionophore, (-)-enniatin B. Tetrahedron Lett. 2012;53:4077–9. https://doi.org/10.1016/j.tetlet.2012.05.110.View ArticleGoogle Scholar
- Lücke D, Dalton T, Ley SV, Wilson ZE. Synthesis of natural and unnatural cyclooligomeric depsipeptides enabled by flow chemistry. Chem (A Eur J). 2016;22:4206–17. https://doi.org/10.1002/chem.201504457.View ArticleGoogle Scholar
- Monma S, Sunazuka T, Nagai K, et al. Verticilide: elucidation of absolute configuration and total synthesis. Org Lett. 2006;8:5601–4. https://doi.org/10.1021/ol0623365.View ArticlePubMedGoogle Scholar
- Batiste SM, Johnston JN. Rapid synthesis of cyclic oligomeric depsipeptides with positional, stereochemical, and macrocycle size distribution control. Proc Natl Acad Sci USA. 2016;113:14893–7. https://doi.org/10.1073/pnas.1616462114.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanaoka M, Isogai A, Suzuki A. Synthesis of bassianolide. Tetrahedron Lett. 1977;18:4049–50. https://doi.org/10.1016/S0040-4039(01)83423-7.View ArticleGoogle Scholar
- Lee H-S, Song H-H, An J-H, et al. Statistical optimization of growth medium for the production of the entomopathogenic and phytotoxic cyclic depsipeptide beauvericin from Fusarium oxysporum KFCC 11363P. J Microbiol Biotechnol. 2008;18:138–44.PubMedGoogle Scholar
- Xu L-J, Liu Y-S, Zhou L-G, Wu J-Y. Enhanced beauvericin production with in situ adsorption in mycelial liquid culture of Fusarium redolens Dzf2. Process Biochem. 2009;44:1063–7. https://doi.org/10.1016/j.procbio.2009.05.004.View ArticleGoogle Scholar
- Xu Y, Orozco R, Wijeratne EMK, et al. Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus Beauveria bassiana. Chem Biol. 2008;15:898–907. https://doi.org/10.1016/j.chembiol.2008.07.011.View ArticlePubMedGoogle Scholar
- Zobel S, Kumpfmüller J, Süssmuth RD, Schweder T. Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin. Appl Microbiol Biotechnol. 2015;99:681–91. https://doi.org/10.1007/s00253-014-6199-0.View ArticlePubMedGoogle Scholar
- Yu D, Xu F, Zi J, et al. Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae. Metab Eng. 2013;18:60–8. https://doi.org/10.1016/j.ymben.2013.04.001.View ArticlePubMedGoogle Scholar
- Meyer V, Andersen MR, Brakhage AA, et al. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biol Biotechnol. 2016;3:6. https://doi.org/10.1186/s40694-016-0024-8.View ArticlePubMedPubMed CentralGoogle Scholar
- Meyer V, Wanka F, van Gent J, et al. Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger. Appl Environ Microbiol. 2011;77:2975–83. https://doi.org/10.1128/AEM.02740-10.View ArticlePubMedPubMed CentralGoogle Scholar
- Richter L, Wanka F, Boecker S, et al. Engineering of Aspergillus niger for the production of secondary metabolites. Fungal Biol Biotechnol. 2014;1:4. https://doi.org/10.1186/s40694-014-0004-9.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee C, Görisch H, Kleinkauf H, Zocher R. A highly specific d-hydroxyisovalerate dehydrogenase from the enniatin producer Fusarium sambucinum. J Biol Chem. 1992;267:11741–4.PubMedGoogle Scholar
- Schuetze T, Meyer V. Polycistronic gene expression in Aspergillus niger. Microb Cell Fact. 2017;16:162. https://doi.org/10.1186/s12934-017-0780-z.View ArticlePubMedPubMed CentralGoogle Scholar
- Mattern IE, van Noort JM, van den Berg P, et al. Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet. 1992;234:332–6.View ArticlePubMedGoogle Scholar
- Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.View ArticlePubMedPubMed CentralGoogle Scholar
- Altschul SF, Wootton JC, Gertz EM, et al. Protein database searches using compositionally adjusted substitution matrices. FEBS J. 2005;272:5101–9. https://doi.org/10.1111/j.1742-4658.2005.04945.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Calvo AM, Wilson RA, Bok JW, Keller NP. Relationship between secondary metabolism and fungal development. Microbiol Mol Biol Rev. 2002;66:447–59. https://doi.org/10.1128/MMBR.66.3.447.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanaoka M, Isogai A, Murakoshi S, et al. Bassianolide, a new insecticidal cyclodepsipeptide from Beauveria bassiana and Verticillium lecanii. Agric Biol Chem. 1978;42:629–35. https://doi.org/10.1271/bbb1961.42.629.Google Scholar
- Monod J. The growth of bacterial cultures. Annu Rev Microbiol. 1949;3:371–94.View ArticleGoogle Scholar
- Kishore G, Sugumaran M, Vaidyanathan CS. Metabolism of dl-(±)-phenylalanine by Aspergillus niger. J Bacteriol. 1976;128:182–91.PubMedPubMed CentralGoogle Scholar
- Schultz JS. Cotton closure as an aeration barrier in shaken flask fermentations. Appl Microbiol. 1964;12:305–10.PubMedPubMed CentralGoogle Scholar
- Krappmann S, Braus GH. Nitrogen metabolism of Aspergillus and its role in pathogenicity. Med Mycol. 2005;43:31–40. https://doi.org/10.1080/13693780400024271.View ArticleGoogle Scholar
- Zhou Z, Takaya N, Nakamura A, et al. Ammonia fermentation, a novel anoxic metabolism of nitrate by fungi. J Biol Chem. 2002;277:1892–6. https://doi.org/10.1074/jbc.M109096200.View ArticlePubMedGoogle Scholar
- Sikora LA, Marzluf GA. Regulation of l-phenylalanine ammonia-lyase by l-phenylalanine and nitrogen in Neurospora crassa. J Bacteriol. 1982;150:1287–91.PubMedPubMed CentralGoogle Scholar
- Vödisch M, Scherlach K, Winkler R, et al. Analysis of the Aspergillus fumigatus proteome reveals metabolic changes and the activation of the pseurotin A biosynthesis gene cluster in response to hypoxia. J Proteome Res. 2011;10:2508–24. https://doi.org/10.1021/pr1012812.View ArticlePubMedPubMed CentralGoogle Scholar
- Barker BM, Kroll K, Vödisch M, et al. Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter. BMC Genom. 2012;13:62. https://doi.org/10.1186/1471-2164-13-62.View ArticleGoogle Scholar
- Kroll K, Pähtz V, Hillmann F, et al. Identification of hypoxia-inducible target genes of Aspergillus fumigatus by transcriptome analysis reveals cellular respiration as an important contributor to hypoxic survival. Eukaryot Cell. 2014;13:1241–53. https://doi.org/10.1128/EC.00084-14.View ArticlePubMedPubMed CentralGoogle Scholar
- Feifel SC, Schmiederer T, Hornbogen T, et al. In vitro synthesis of new enniatins: probing the alpha-d-hydroxy carboxylic acid binding pocket of the multienzyme enniatin synthetase. ChemBioChem. 2007;8:1767–70. https://doi.org/10.1002/cbic.200700377.View ArticlePubMedGoogle Scholar
- Matthes D, Richter L, Müller J, et al. In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues. Chem Commun. 2012;48:5674–6. https://doi.org/10.1039/c2cc31669b.View ArticleGoogle Scholar
- Müller J, Feifel SC, Schmiederer T, et al. In vitro synthesis of new cyclodepsipeptides of the PF1022-type: probing the alpha-d-hydroxy acid tolerance of PF1022 synthetase. ChemBioChem. 2009;10:323–8. https://doi.org/10.1002/cbic.200800539.View ArticlePubMedGoogle Scholar
- Xu Y, Wijeratne EMK, Espinosa-Artiles P, et al. Combinatorial mutasynthesis of scrambled beauvericins, cyclooligomer depsipeptide cell migration inhibitors from Beauveria bassiana. ChemBioChem. 2009;10:345–54. https://doi.org/10.1002/cbic.200800570.View ArticlePubMedGoogle Scholar
- Boecker S, Zobel S, Meyer V, Süssmuth RD. Rational biosynthetic approaches for the production of new-to-nature compounds in fungi. Fungal Genet Biol. 2016;89:89–101. https://doi.org/10.1016/j.fgb.2016.02.003.View ArticlePubMedGoogle Scholar
- Huddleston JP, Johnson WH, Schroeder GK, Whitman CP. Reactions of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue, with acetylene and allene substrates: evidence for a hydration-dependent decarboxylation. Biochemistry. 2015;54:3009–23. https://doi.org/10.1021/acs.biochem.5b00240.View ArticlePubMedPubMed CentralGoogle Scholar
- EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific opinion on the risks to human and animal health related to the presence of beauvericin and enniatins in food and feed. EFSA J. 2014;12:3802. https://doi.org/10.2903/j.efsa.2014.3802.View ArticleGoogle Scholar
- European Food Safety Authority. Occurrence data on beauvericin and enniatins in food. 2017. https://doi.org/10.5281/ZENODO.571179.Google Scholar
- Jestoi M. Emerging Fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin: a review. Crit Rev Food Sci Nutr. 2008;48:21–49. https://doi.org/10.1080/10408390601062021.View ArticlePubMedGoogle Scholar
- Santini A, Ferracane R, Meca G, Ritieni A. Overview of analytical methods for beauvericin and fusaproliferin in food matrices. Anal Bioanal Chem. 2009;395:1253–60. https://doi.org/10.1007/s00216-009-3117-x.View ArticlePubMedGoogle Scholar
- Sørensen JL, Nielsen KF, Rasmussen PH, Thrane U. Development of a LC-MS/MS method for the analysis of enniatins and beauvericin in whole fresh and ensiled maize. J Agric Food Chem. 2008;56:10439–43. https://doi.org/10.1021/jf802038b.View ArticlePubMedGoogle Scholar
- Uhlig S, Ivanova L. Determination of beauvericin and four other enniatins in grain by liquid chromatography-mass spectrometry. J Chromatogr A. 2004;1050:173–8. https://doi.org/10.1016/j.chroma.2004.08.031.View ArticlePubMedGoogle Scholar
- Sewram V, Nieuwoudt TW, Marasas WF, et al. Determination of the Fusarium mycotoxins, fusaproliferin and beauvericin by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr A. 1999;858:175–85.View ArticlePubMedGoogle Scholar
- Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003;49:1041–4. https://doi.org/10.1373/49.7.1041.View ArticlePubMedGoogle Scholar
- Panuwet P, Hunter RE, D’Souza PE, et al. Biological matrix effects in quantitative tandem mass spectrometry-based analytical methods: advancing biomonitoring. Crit Rev Anal Chem. 2016;46:93–105. https://doi.org/10.1080/10408347.2014.980775.View ArticlePubMedPubMed CentralGoogle Scholar
- Berg T, Strand DH. 13C labelled internal standards-A solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples? J Chromatogr A. 2011;1218:9366–74. https://doi.org/10.1016/j.chroma.2011.10.081.View ArticlePubMedGoogle Scholar
- Varga E, Glauner T, Köppen R, et al. Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS. Anal Bioanal Chem. 2012;402:2675–86. https://doi.org/10.1007/s00216-012-5757-5.View ArticlePubMedPubMed CentralGoogle Scholar
- Rychlik M, Asam S. Stable isotope dilution assays in mycotoxin analysis. Anal Bioanal Chem. 2008;390:617–28. https://doi.org/10.1007/s00216-007-1717-x.View ArticlePubMedGoogle Scholar
- Hu L, Rychlik M. Biosynthesis of 15N3-labeled enniatins and beauvericin and their application to stable isotope dilution assays. J Agric Food Chem. 2012;60:7129–36. https://doi.org/10.1021/jf3015602.View ArticlePubMedGoogle Scholar
- Stokvis E, Rosing H, Beijnen JH. Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun Mass Spectrom. 2005;19:401–7. https://doi.org/10.1002/rcm.1790.View ArticlePubMedGoogle Scholar
- Boyce M, Monnot F. First in man clinical trial of emodepside (BAY 44-4400). In: Clin. NCT02661178. 2017. https://clinicaltrials.gov/show/NCT02661178. Accessed 25 Oct 2017.
- Sambrook J, Russel DW. Molecular cloning—a laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.Google Scholar
- Meyer V, Ram AF, Punt PJ. Genetics, genetic manipulation, and approaches to strain improvement of filamentous fungi. In: Baltz RH, Davies JE, Demain AL, editors. Man. Ind. Microbiol. Biotechnol. 3rd ed. American Society for Microbiology (ASM); 2010. pp 318–30.Google Scholar
- Vishniac W, Santer M. The thiobacilli. Microbiol Mol Biol Rev. 1957;21:195–213.Google Scholar
- Iversen JJL, Thomsen JK, Cox RP. On-line growth measurements in bioreactors by titrating metabolic proton exchange. Appl Microbiol Biotechnol. 1994;42:256–62. https://doi.org/10.1007/BF00902726.View ArticleGoogle Scholar
- Baldwin JE, Adlington RM, Crouch NP, Pereira IAC. The enzymatic synthesis of isotopically labelled penicillin Ns with isopenicillin N synthase. J Label Compd Radiopharm. 1998;41:1145–63. https://doi.org/10.1002/(SICI)1099-1344(199812)41:12<1145::AID-JLCR159>3.0.CO;2-2.
- Madry N, Zocher R, Kleinkauf H. Enniatin production by Fusarium oxysporum in chemically defined media. Appl Microbiol Biotechnol. 1983;17:75–9.View ArticleGoogle Scholar