Huesemann, L. | Search bioRxiv

bioRxiv2023

Engineering and implementation of synthetic molecular tools in the basidiomycete fungus Ustilago maydis

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. We generated dual-reporter constructs 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, 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 in U. maydis and enable in planta detection of fungal infection.

microbiology↗

bioRxiv2020

Polycistronic gene expression in the model microorganism Ustilago maydis

AbtractEukaryotic microorganisms transcribe 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. 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.

microbiology↗