Correction to: Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

[This corrects the article DOI: 10.1186/s40694-018-0048-3.].

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Mouse tissue distribution and persistence of the food-born fusariotoxins Enniatin B and Beauvericin.

The fusariotoxins Enniatin B (Enn B) and Beauvericin (Bea) have recently aroused interest as food contaminants and as potential anticancer drugs. However, limited data are available about their toxic profile. Aim of this study was to investigate their pharmacological behavior in vivo and their persistence in mice. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the distribution of Enn B and Bea in selected tissue samples and biological fluids originating from mice treated intraperitoneally with these cyclohexadepsipeptides. Overall, no toxicological signs during life time or pathological changes were observed. Moreover, both fusariotoxins were found in all tissues and serum but not in urine. Highest amounts were measured in liver and fat demonstrating the molecules tendency to bioaccumulate in lipophilic tissues. While for Bea no metabolites could be detected, for Enn B three phase I metabolites (dioxygenated-Enn B, mono- and di-demethylated-Enn B) were found in liver and colon, with dioxygenated-Enn B being most prominent. Consequently, contribution of hepatic as well as intestinal metabolism seems to be involved in the overall metabolism of Enn B. Thus, despite their structural similarity, the metabolism of Enn B and Bea shows distinct discrepancies which might affect long-term effects and tolerability in humans.

The naturally born fusariotoxin enniatin B and sorafenib exert synergistic activity against cervical cancer in vitro and in vivo.

During the last decades substantial progress has been made in developing systemic cancer therapy. However, tumors are frequently intrinsically resistant against structurally and mechanistically unrelated drugs. Thus, it is of predominant interest to overcome drug resistance and to encourage the research for novel chemotherapeutic approaches. Recently, we have introduced enniatins, naturally occurring cyclohexadepsipeptides produced by filamentous fungi of the genus Fusarium, as potential anticancer drugs. Here, we expend this approach by demonstrating antiangiogenic properties for enniatin B (Enn B) indicated by a strong inhibition of human endothelial cell migration and tube formation. Moreover, combination of Enn B with the clinically approved multi-kinase inhibitor sorafenib (Sora) displayed profound synergistic in vitro and in vivo anticancer effects against cervical cancer. Subsequent studies showed that this strong synergism is accompanied by a marked increase in mitochondrial injury and apoptosis induction reflected by mitochondrial membrane depolarization, caspase-7 activation, and subsequent cleavage of PARP. Additionally, cells were shown to stop DNA synthesis and accumulate in S and G2/M phase of the cell cycle. The multifaceted characteristics underlying this strong synergism were suggested to be based on interference with the p38 MAPK as well as the ERK signaling pathways. Finally, also in vivo studies revealed that the combination treatment is distinctly superior to single drug treatments against the KB-3-1 cervix carcinoma xenograft model. Taken together, our data confirm the anticancer benefits of the naturally occurring fusariotoxin Enn B and further present Enn B/Sora as a novel combination strategy especially for the treatment of cervical cancer.

Engineering of Aspergillus niger for the production of secondary metabolites.

BACKGROUND: Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l-1) of secondary metabolites belonging to the class of nonribosomal peptides. RESULTS: For a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l-1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv. CONCLUSIONS: This is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.

In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues.

New beauvericins have been synthesized using the nonribosomal peptide synthetase BbBEAS from the entomopathogenic fungus Beauveria bassiana. Chemical diversity was generated by in vitro chemoenzymatic and in vivo whole cell biocatalytic syntheses using either a B. bassiana mutant or an E. coli strain expressing the bbBeas gene.