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

Abstract

BackgroundFungal 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.ResultsThe 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.ConclusionIn 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.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0048-3) contains supplementary material, which is available to authorized users.

Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites

“…This finding proved that polycistronic secondary metabolite biosynthesis is possible in A. niger and suggested that the KivR enzyme catalyses the rate-limiting step in enniatin B biosynthesis [ 11 ]. It was furthermore demonstrated that A. niger is (i) a superior expression host not only for enniatin B, but also for beauvericin and bassianolide, by producing the highest titres ever reported for heterologous hosts as well as for natural producing organisms [ 12 ]; and (ii) an ideal platform strain for the production of new-to-nature CDPs obtained by designing and expressing hybrid CDP synthetases in A. niger [ 13 , 14 ]. Indeed, some of the novel CDPs displayed considerably higher bioactivities compared to their parental CDPs and reference drugs [ 14 ].…”

Fungal Biotechnology in Space: Why and How?

“…A. niger's metabolic diversity has been expanded to overproduce the secondary metabolites enniatin, bassianolide and beauvericin with high pharmaceutical application potential due to their antibacterial, antifungal and anticancer activities (Boecker et al 2018). Hence, A. niger is a powerful multipurpose cell factory producing a wide range of products, which-in such a diversity and efficiencyare not matched by any other fungal cell factory.…”

Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production

“…The esyn1 NRPS gene from Fusarium oxysporum was expressed in A. niger, leading to the production of enniatin at initial un-optimized titer of 1 mg.l -1 (Richter et al, 2014). Beauvericin and bassianolide were also heterologously produced after the cloning of the corresponding bbBeas and bbBsls synthetase genes from B. bassiana (Boecker et al, 2018). In these two studies, a promising A. niger heterologous expression host was developed based on the Tet-on bacterial-fungal hybrid expression system.…”

“…The expression strength is directly correlated to the Dox concentration added, thus making Tet-on tuneable and very efficient, with expression levels equivalent to those obtained with the widely used strong constitutive promoter P gpdA (Meyer et al, 2011). In addition, the impact of the number of bbBeas gene copies integrated in A. niger genome on the beauvericin productivity was also investigated (Boecker et al, 2018). The strain harbouring a tandem gene copy produced approximately two-fold higher titers of beauvericin than the strain harbouring a single copy, under similar culture conditions (628 versus 294 mg•l -1 ).…”