Eukaryotic microorganisms use monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. During translation of these peptides the ribosome stalls, the peptide chain is released and the ribosome resumes translation. Thus, two independent polypeptide chains can be encoded from a single mRNA when a 2A peptide sequence is placed inbetween the two open reading frames. Here, we establish such a system in the well-studied model microorganism
Ustilago maydis
. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated
in vivo
using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides
in vivo
and demonstrated the applicability of 2A peptide technology for
U. maydis
in basic and applied science.
The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen–host interactions and of great interest for a broad spectrum of biotechnological applications. To facilitate research and enable applications, in this study, three luminescence-based and one enzymatic quantitative reporter were implemented and characterized. Several dual-reporter constructs were generated for ratiometric normalization that can be used as a fast-screening platform for reporter gene expression, applicable to in vitro and in vivo detection. Furthermore, synthetic bidirectional promoters that enable bicisitronic expression for gene expression studies and engineering strategies were constructed and implemented. These noninvasive, quantitative reporters and expression tools will significantly widen the application range of biotechnology in U. maydis and enable the in planta detection of fungal infection.
The basidiomyceteUstilago maydisis a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. To facilitate research and enable applications, in this study, three luminescence-based and one enzymatic quantitative reporter were implemented and characterized. We generated dual-reporter constructs for ratiometric normalization that can be used as a fast-screening platform for reporter gene expression, applicable toin vitroandin vivodetection. Furthermore, we constructed and implemented synthetic bi-directional promoters, that enable bicisitronic expression for gene expression studies and engineering strategies. These non-invasive, quantitative reporters and expression tools will widen significantly the application range of biotechnology inU. maydisand enable in planta detection of fungal infection.
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