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Polycistronic gene expression in Aspergillus niger.

Tabea Schuetze1, Vera Meyer2

  • 1Department of Applied Microbiology and Genetics, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany. tabea.schuetze@tu-berlin.de.

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|September 27, 2017
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Summary

The viral P2A peptide enables polycistronic gene expression in Aspergillus niger, facilitating the activation of cryptic fungal secondary metabolite gene clusters. This method allows for tunable expression of multiple genes from a single cassette.

Keywords:
Aspergillus nigerEnniatin BHeterologous gene expressionLuciferaseP2A peptidePolycistronicSecondary metabolite

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Area of Science:

  • Molecular Biology
  • Mycology
  • Metabolic Engineering

Background:

  • Filamentous fungi possess numerous predicted biosynthetic gene clusters, many of which remain unexpressed ('cryptic') in their native hosts.
  • Heterologous expression is a key strategy to activate these gene clusters and discover novel bioactive compounds.
  • Polycistronic expression offers a streamlined approach for co-expressing multiple genes from a single tunable promoter, simplifying cloning and controlling gene expression.

Purpose of the Study:

  • To evaluate the efficacy of the viral P2A peptide for co-expressing multiple genes in the filamentous fungus Aspergillus niger.
  • To assess the impact of gene positioning within a polycistronic cassette on expression levels.
  • To enable the heterologous expression of fungal secondary metabolite gene clusters.

Main Methods:

  • Constructed three polycistronic expression cassettes utilizing the P2A peptide for co-expression of two enniatin biosynthesis genes (esyn1, ekivR) and a luciferase reporter gene (luc) in A. niger.
  • Integrated these cassettes into the pyrG locus of A. niger.
  • Employed a Tet-On system for tunable, doxycycline-inducible expression.
  • Analyzed gene expression via Northern blotting and quantified luciferase activity to determine position effects.

Main Results:

  • Successfully demonstrated polycistronic expression of three genes in A. niger, confirmed by the production of a single polycistronic mRNA.
  • Achieved comparable production of the secondary metabolite enniatin across all tested strains, irrespective of the luciferase gene's position.
  • Observed significant gene position effects on luciferase activity, with the lowest activity at the first position and comparable levels at the second and third positions.

Conclusions:

  • The viral P2A peptide is a viable tool for achieving polycistronic gene expression in Aspergillus niger.
  • This method facilitates the co-expression of multiple genes, including entire secondary metabolite gene clusters, in equimolar amounts.
  • The findings provide a foundation for activating and characterizing cryptic fungal natural products through heterologous expression.