Abstract
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.
Highlights
Gene expression is structured in operons containing polycistronic mRNAs encoding multiple proteins
To test the activity of different 2A peptides in U. maydis, we designed a bi-cistronic reporter system consisting of the following components (Figure 1A): (i) constitutively active promoter, (ii) upstream ORF encoding a red fluorescent protein, (iii) 2A peptide of interest, (iv) downstream ORF encoding a green fluorescent protein fused to a nuclear localisation signal (NLS) and (v) heterologous transcriptional terminator
An active 2A peptide would result in increased cytoplasmic red fluorescence while green fluorescence will be located in the nucleus
Summary
Gene expression is structured in operons containing polycistronic mRNAs encoding multiple proteins This has the clear advantage that expression of several proteins can be regulated synchronously using a single promoter and terminator. For genetic and metabolic engineering it is advantageous to mimic polycistronic mRNAs in eukaryotes for efficient co-regulation of mRNAs in a defined spatio-temporal manner. This circumvents, for example, the multiple uses of identical promoters and terminators, which might reduce overall promoter activity or could interfere with strain generation using homologous recombination (de Felipe et al, 2006; Unkles et al, 2014)
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