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Main Title: Engineering of Aspergillus niger for the production of secondary metabolites
Author(s): Richter, Lennart
Wanka, Franziska
Boecker, Simon
Storm, Dirk
Kurt, Tutku
Vural, Özlem
Süßmuth, Roderich D.
Meyer, Vera
Type: Article
Language Code: en
Is Part Of: 10.14279/depositonce-5419
Abstract: 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 ɑ-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.
Issue Date: 2014
Date Available: 16-Aug-2016
DDC Class: 570 Biowissenschaften; Biologie
Subject(s): Aspergillus niger
secondary metabolite
nonribosomal peptide synthetase
heterologous gene expression
nonribosomale Peptidsynthetase
heterologe Expression
Sponsor/Funder: EC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QuantFung
DFG, EXC 314, Unifying Concepts in Catalysis
Journal Title: Fungal Biology and Biotechnology
Publisher: BioMed Central
Publisher Place: London
Volume: 1
Article Number: 4
Publisher DOI: 10.1186/s40694-014-0004-9
EISSN: 2054-3085
Appears in Collections:FG Angewandte und Molekulare Mikrobiologie » Publications

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