Generating an orthogonal translation compartment in yeast

Lead Research Organisation: University of Aberdeen
Department Name: Institute of Medical Sciences

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M010996/1 01/10/2015 30/09/2023
1654590 Studentship BB/M010996/1 01/10/2015 30/09/2019 Svetlozara Petrova Chobanova
 
Description During the course of my PhD so far a have discovered two key aspects, which are crucial, for the synthetic mechanism I am trying to create. I have shown that a specific translation intiation factor can be used to direct translation initiaton in my organism of study. In the same organism, I have engeneered a trans-splicing reaction, which is the base for the building on my syhthetic construct.
Exploitation Route It could potentially provide a novel mechanism for improved protein production.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title Produced yeast, which is capable of trans-splicing. 
Description By engeneering S. cerevisiae cells to express contructs, which produce elements required for a sucsessfull trans-splicing reaction, I was eble to convey yeast cells to trans-splice. This has not been reported previously. The engeneering of the trans-splcing reaction is a novel approach, which also has not been previosuly reported. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? No  
Impact Made a huge impact over the outcome of my PhD project and could pontentilly lead to developing a novel mechamism for recombinant protein production. 
 
Description British Science Week event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I took part in a live 'Speed Science' event, which is part of the British Science Week activities in the partner University of Aberdeen on the 17-18 March 2016. Event Description (Taken from the Britsh Science Week page) : "Like speed dating, without the dating! Join the brightest stars in Scottish biology for a scintillating 'speed science' session where you, the audience, get to hear about the latest research from PhD students working at its forefront. But can they keep you interested and wanting to know more? Come and vote on the best of the biology bunch at this fast paced event hosted by the University of Aberdeen's Public Engagement team in partnership with EASTBIO - the East of Scotland Biological Science Doctoral Training Partnership."
Year(s) Of Engagement Activity 2016
URL https://www.abdn.ac.uk/engage/nsew/index.php#thur17