Generating an orthogonal translation compartment in yeast

Saccharomyces cerevisiae is an important organism used for the production of medically and commercially important biomolecules in industrial scale processes in pharmaceutical and biotechnology industries. A major challenge in the use of S. cerevisiae in these applications is to maximise the production of heterologously expressed molecules whilst minimising the impact of this expression on the metabolism and other cellular activities of the cell. The project will focus on achieving this through the 'compartmentalisation' of the translation machinery by generating mRNAs that are recognised by specialised, orthogonal translation initiation factors.

Translation initiation in eukaryotes is the key regulatory step in translation, and this event is dependent on the interaction between the 5' modification of the mRNA, the cap, and the eIF4 initiation factor. In nematodes, a substantial fraction of mRNAs are 'trans-spliced' to a set of short, 'spliced leader' (SL) RNAs. Since these SL RNAs are modified at their 5'-ends with a trimethyl guanosine cap, instead of the conventional monomethyl guanosine cap of most mRNAs, this modified cap is present at the 5' ends of all trans-spliced mRNAs. Because of their different cap structure SL trans-spliced mRNAs are poorly recognised by most eIF4 proteins, and nematodes have consequently evolved eIF4 isoforms that have higher affinities for trimethyl guanosine capped mRNAs.

SL trans-splicing is sporadically distributed across the eukaryotes, and has not been reported in fungi. The project will engineer yeast cells with the ability to add spliced leaders onto the 5'-ends of specific mRNAs, coupled with expression of trimethyl guanosine-SL-specific eIF4 factors. This will allow us to control translation initiation at the level of individual mRNA species and potentially reduce the competition between translation initiation of the heterologous mRNA and the endogenous yeast mRNAs.