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Applications of yeast display in synthetic biology with a focus on biological synthesis and recycling of plastics
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Date
2025
Authors
Jurić, Vanja
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Publisher
University College Cork
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Abstract
Yeast surface display has emerged as a versatile tool in synthetic biology, particularly in protein engineering. This work sought to improve the flexibility and ease-of-use of yeast display for synthetic biologists, as well as applying the yeast display system in several contexts – with a particular focus on biological synthesis and recycling of plastics.
The yeast modular cloning toolkit (MoClo-YTK) utilizes type IIS restriction enzymes for the hierarchical assembly of expression vectors from standardised parts into Saccharomyces cerevisiae expression cassettes. In Chapter 3, to facilitate the use of this toolkit for yeast surface display experiments, parts that allow for the incorporation of five surface display anchor proteins with various epitope tags into MoClo-YTK-compatible expression constructs were created. Building on prior work in the Young group allows these surface anchor proteins to be combined with a library of signal peptides (SPs) and translational fusion partners (TFPs) to optimise surface display of a protein of interest – a significant challenge for many recombinant proteins. This expansion of the MoClo-YTK yeast was validated using several proteins of interest and constitutes a yeast secretion/display toolkit (MoClo-YSD) that has been made available to the research community.
The MoClo-YSD toolkit allowed the efficient use of yeast display for a range of different projects. It was successfully applied to develop a new approach to co-immunoprecipitation, where instead of immobilising an antibody on agarose beads, the antibody is displayed on the yeast surface, effectively using yeast as beads. As a proof of concept, an anti-GFP nanobody was used to capture GFP from bacterial lysates and GFP-tagged LNX1 protein from mammalian cell lysates. Co-precipitation of the LNX1 binding partners liprin-α1 and Numb, showed that yeast display can be used for studying protein-protein interactions. Furthermore, in Chapter 4, the MoClo YSD toolkit was adapted to co-display enzymes capable of degrading polyethylene terephthalate (PET), a traditional plastic that presents an environmental challenge due to its non-biodegradable nature and the resultant microplastic pollution. Enzymes such as LC-cutinase and MHETase, as well as hydrophobin, a protein that facilitates yeast attachment to hydrophobic surfaces, were successfully co-displayed, achieving notable degradation of low-crystallinity PET.
Additionally, in Chapter 5, a method for developing biosensors for small molecules utilising yeast display was tested in this work. One application of such biosensors would be the detection of metabolites of interest during strain development and optimization for the production of biobased plastics. The biosensor design strategy employed yeast display with the goal of identifying protein-protein interactions that can be disrupted by the target small molecule, and thereafter adapted into a working biosensor. While a nanobody library was successfully screened to select for nanobodies that bind the target proteins (small molecule binders Dig10.2 and FDCA decarboxylase) the disruption of the nanobody-protein interactions by the small molecule analytes has not yet been demonstrated. However, nanobodies that specifically bind Dig10.2 were expressed in bacteria, purified and characterised in a plate-based binding assay and may be useful in other lines of research.
Overall, this work highlights the diverse applications of yeast display in synthetic biology, from studying protein-protein interactions, to enhancing the binding of an enzyme to its substrate, environmental solutions and biosensing technologies.
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Keywords
Yeast , Yeast display , Nanobody , PET , Polyethylene terephthalate , Plastics , Saccharomyces cerevisiae , Recombinant protein production , Immunoprecipitation
Citation
Jurić, V. 2025. Applications of yeast display in synthetic biology with a focus on biological synthesis and recycling of plastics. PhD Thesis, University College Cork.