Engineering of 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 to produce high titers (up to 4,500 mg • l) 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 in . ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of 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 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating in 5 l fed batch fermentations. Finally, an autonomous 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 -expressing strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv.

Conclusions: This is the first report demonstrating that 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 allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.