Producing Hydrophobins by Increasing Secretion and SUMOylation in Yarrowia Lipolytica

• Shirly Gottlieb, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical & Biomolecular Engineering, University of Delaware

Hydrophobins are proteins active at the surface and produced solely by fungi. They self-assemble at their interface and have the ability to turn previously hydrophilic structures into hydrophobic shapes, and vice versa. Hydrophobins are beneficial for coatings, biocatalysts, and recombinant protein purification. The yeast strain Yarrowia lipolytica can be engineered to increase secretion and express these proteins at a high yield. Furthermore, to increase protein solubility and expression of the hydrophobins, SUMO (Small Ubiquitin-Like Modifiers) tags, which have been proven to increase solubility, can be fused to the hydrophobin gene. Several purification methods were attempted and then optimized to attempt to purify the proteins such as a methanol-chloroform protocol, pull-down assay, and the use of various high-tech centrifuges. A simple cloning was performed to fuse the SUMO tag to the recombinant protein. While the results from the purification methods were not entirely conclusive, there remains significant outcomes from performing gel electrophoresis that may suggest there are polymerized hydrophobins in the Y. Lipolytica strains. This allows for advancing the production of hydrophobins in yeast and maximizing the purification methods. Further research and the enhancement of purification methods can help obtain higher yields of hydrophobins as well as improve the secretion in Yarrowia lipolytica for the use of various proteins beyond hydrophobins.