Researcher(s)
- Lauren DeSantis, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
- , , University of Delaware
Abstract
A desirable outcome in the field of pharmaceuticals is increasing production efficiency of plant based medicines. Yarrowia lipolytica is an unconventional but desirable yeast due to its oleaginous phenotype, which lends itself to natural product biosynthesis. While the industry is usually limited to model yeasts, such as baker’s yeast, S. cerevisiae for heterologous production of complex natural products, our goal is to bioengineer Y. lipolytica to biosynthesize strictosidine, a gateway molecule to thousands of plant based medicines . By bioengineering our chosen yeast and optimizing production conditions, we will be working towards a cheaper and more robust supply of plant-based cancer therapeutics.
Standard molecular biology techniques are utilized in this project, such as polymerase chain reactions to amplify genes of interest and Hifi assemblies as well as golden gate reactions to assemble them. Our main methods of verifying proper cloning is through gel electrophoresis, restriction enzyme digestions, and DNA sequencing.
The strictosidine pathway is long, with over 13 genes needing to be integrated. To integrate larger pieces of the pathway in fewer steps, we will use a serine integrase, an enzyme derived from a bacteriophage. A small landing pad, recognized by the integrase, will be randomly integrated in the yeast using a native mechanism, and will be used to integrate the genes of interest into Y. lipolytica. Furthermore, we will make use of fluorescent proteins to determine integration efficiencies by using flow cytometry. Finally, we will be localizing some enzymes in the strictosidine pathway to certain organelles in Y. lipolytica using 3 GFP localization tags.