Engineered Saccharomyces cerevisiae as a biosynthetic platform of nucleotide sugars and heterologous glycosylation
- Crowe, Samantha Aiko
- Advisor(s): Keasling, Jay D
Abstract
Glycosylation is a vital modification found all over biology. It can affect both structure and function of biomolecules as well as solubility and stability. Despite its ubiquity, glycosylation is still vastly understudied. This is in large part due to the lack of availability of glycosylation precursors which mostly comprise nucleotide sugars. Saccharomyces cerevisiae is a powerful platform that has been used to produce a wide variety of molecules for both scale up and elucidation of plant natural product pathways, however, S. cerevisiae lacks nucleotide sugar diversity. Nucleotide sugars are synthesized primarily two different ways either by the salvage pathway starting from a specific sugar or de novo synthesis which interconverts nucleotide sugars, typically starting from Uridine-diphosphate-D-glucose. This metabolism is complicated with strong feedback mechanisms and reactions governed by thermodynamic equilibriums. Here I present our work in engineering yeast for complex glycosylations with greater nucleotide sugar diversity. I engineered yeast to make 7 heterologous nucleotide sugars, studying how these pathways interact with each other and mitigating different challenges in nucleotide sugar production. We then engineered yeast to glycosylated and synthesize QS-21, a desired vaccine-adjuvant saponin that contains 6-7 unique sugars. This work serves as a foundation for a much greater diversity of possible glycosylated secondary metabolites and proteins to be made in yeast.
Chapter 1 provides a background on nucleotide sugar synthesis, metabolism, as well as glycosylation engineering in Saccharomyces cerevisiae. Chapter 2 details the development of S. cerevisiae as a platform for producing nucleotide sugars and studying their metabolism. Chapter 3 describes our work in the glycosylation and complete biosynthesis of QS-21, a desired vaccine adjuvant in engineered yeast, which contains 6-7 unique sugars. Chapter 4 is a short conclusion chapter with reflections and future perspectives in this field of research.