Researcher(s)
- Shirly Gottlieb, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Yarrowia lipolytica is a GRAS non-conventional yeast known for its high protein secretion efficiency. The secretory capabilities of Y. lipolytica can be utilized through a yeast surface display (YSD) experiment. In this technique, proteins of interest travel through the secretory pathway, but instead of being secreted out of the cell, are anchored to the yeast cell wall, enhancing stability against environmental changes. The surface display platform can be used to create microbial biosensors, improve substrate availability, and for high-throughput screening. In this study, the aim is to employ a CRISPR knockout library in Y. lipolytica to increase its secretion capabilities. As a proof of concept, an anti-GFP nanobody was tethered to the yeast cell surface using a CWP–GPI (Cell Wall Protein–Glycosylphosphatidylinositol) anchor. Cells were incubated with GFP and analyzed via flow cytometry to confirm successful surface display of the nanobody. It was hypothesized that the nanobody was positioned too close to the cell surface for effective display. Therefore, to connect the anti-GFP nanobody to the CWP-GPI anchor, five peptide linkers were cloned: three differently sized linkers made of glycine and serine were tested to introduce flexibility and two linkers: one composed of alanine and proline amino acids, and the other of glutamic acid, alanine, and lysine were tested for their rigidity. Flow cytometry was then used to evaluate the surface display efficiency of each construct. Results indicated that the linkers did not have a significant effect on surface display abilities and further testing is required such as alternative linkers, cleavable fluorescent tags, and multi-copy integration.