Abstract
BackgroundGenome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger.ResultsIn this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three.ConclusionsThe P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.
Highlights
Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far
Our results show that polycistronic gene expression did allow expression of all three proteins and the successful production of enniatin
In this study, we proved that the P2A-mediated polycistronic gene expression is possible in A. niger
Summary
Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. Polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. Beekwilder et al established it for the yeast Saccharomyces cerevisiae to produce β-carotene and Unkles et al were able to express the penicillin gene cluster, encoding the ACV synthetase (pcbAB), isopenicillin N synthase (pcbC) and isopenicillin N acyltransferase (penDE) in the filamentous fungus Aspergillus nidulans using the P2A peptide [10, 11]. The use of the viral 2A peptide for polycistronic gene expression in eukaryotes was first described in 2004 [12], where three different 2A sequences (E2A—equine rhinitis A virus, F2A—foot-andmouth disease virus and T2A—Thoseaasigna virus) were used to assemble four transmembrane proteins of the T-cell receptor CD3 complex
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