Blog
About

  • Record: found
  • Abstract: found
  • Article: found
Is Open Access

Engineering of Aspergillus niger for the production of secondary metabolites

Read this article at

Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      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.

      Related collections

      Most cited references 58

      • Record: found
      • Abstract: found
      • Article: not found

      Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88.

      The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        Regulation of fungal secondary metabolism.

        Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          Fungal secondary metabolites - strategies to activate silent gene clusters.

          Filamentous fungi produce a multitude of low molecular weight bioactive compounds. The increasing number of fungal genome sequences impressively demonstrated that their biosynthetic potential is far from being exploited. In fungi, the genes required for the biosynthesis of a secondary metabolite are clustered. Many of these bioinformatically newly discovered secondary metabolism gene clusters are silent under standard laboratory conditions. Consequently, no product can be found. This review summarizes the current strategies that have been successfully applied during the last years to activate these silent gene clusters in filamentous fungi, especially in the genus Aspergillus. The techniques take advantage of genome mining, vary from the simple search for compounds with bioinformatically predicted physicochemical properties up to methods that exploit a probable interaction of microorganisms. Until now, the majority of successful approaches have been based on molecular biology like the generation of gene "knock outs", promoter exchange, overexpression of transcription factors or other pleiotropic regulators. Moreover, strategies based on epigenetics opened a new avenue for the elucidation of the regulation of secondary metabolite formation and will certainly continue to play a significant role for the elucidation of cryptic natural products. The conditions under which a given gene cluster is naturally expressed are largely unknown. One technique is to attempt to simulate the natural habitat by co-cultivation of microorganisms from the same ecosystem. This has already led to the activation of silent gene clusters and the identification of novel compounds in Aspergillus nidulans. These simulation strategies will help discover new natural products in the future, and may also provide fundamental new insights into microbial communication. Copyright © 2010 Elsevier Inc. All rights reserved.
            Bookmark

            Author and article information

            Affiliations
            [1 ]GRID grid.6734.6, ISNI 0000000122928254, Institute of Chemistry, Department of Biological Chemistry, , Berlin University of Technology, ; Straße des 17, Juni 124, Berlin, 10623 Germany
            [2 ]GRID grid.6734.6, ISNI 0000000122928254, Institute of Biotechnology, Department Applied and Molecular Microbiology, , Berlin University of Technology, ; Gustav-Meyer-Allee 25, Berlin, 13355 Germany
            Contributors
            lennart.richter@chem.tu-berlin.de
            franziska.wanka@tu-berlin.de
            simon.boecker@chem.tu-berlin.de
            dirk@ferbau.de
            t_kurt@gmx.net
            ozlem.vural@gmx.de
            suessmuth@chem.tu-berlin.de
            vera.meyer@tu-berlin.de
            Journal
            Fungal Biol Biotechnol
            Fungal Biol Biotechnol
            Fungal Biology and Biotechnology
            BioMed Central (London )
            2054-3085
            14 October 2014
            14 October 2014
            2014
            : 1
            5598268
            4
            10.1186/s40694-014-0004-9
            © Richter et al.; licensee BioMed Central 2014

            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

            Categories
            Research
            Custom metadata
            © The Author(s) 2014

            Comments

            Comment on this article