Terms of Useengineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.-3 -

BackgroundFilamentous 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.ResultsFor 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40694-014-0004-9) contains supplementary material, which is available to authorized users.

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Non-ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa- and cyclo-octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy-acid-activating module of PF1022 synthetase with the amino-acid-activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.

The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized by Escherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F1‐part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth‐arrested cells, which is vital for its use in two‐stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW−1 h−1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth‐coupled processes) as well as volumetric productivity (in two‐stage processes) depending on which of the performance measures is more crucial for the process and product of interest.