(553d) Engineering Nucleotide Sugar Metabolism in S. Cerevisiae for the Glycosylation of Complex Natural Products and Heterologous Proteins

Glycosylation is an important modification step in cells that affects the bioactivity and stability of secondary metabolites, as well as the localization and function of proteins. S. cerevisiae has the proven ability to produce complex heterologous products and proteins but lacks the necessary precursors for glycosylation because yeast natively only produces a few of them including UDP-glucose and UDP-galactose. Nucleotide sugars in the form of UDP-sugars are the most common substrates for glycosylation in plants, the microbial biosynthesis of which can be achieved via the expression of nucleotide sugar interconversion enzymes (NSEs) that are native of plants. As such, UDP-glucose can also be converted to UDP-glucuronic acid, UDP-galactose, UDP-rhamnose, etc. in yeast. However, nucleotide sugar metabolism is highly regulated by many UDP-sugars inhibiting different NSEs, with UDP-xylose being a particularly important inhibitor of many enzymes involved in nucleotide sugar synthesis in order to maintain a high level of UDP-glucose in the cell. In this work, the heterologous production of various nucleotide sugars in S. cerevisiae as well as effective means to control their relative production levels with different promoters driving the expression of NSEs, as well as metabolic efforts to alleviate the inhibitory feedback caused by UDP-xylose. This work will pave the way for engineering S. cerevisiae to produce bioactive and complex glycosylated products with importance in food and pharmaceutical products.