(160ao) Nucleotide Sugar Production By Engineered Yeast

Nucleotide sugars are activated monosaccharides with many applications. These compounds play a role as intermediates in glycosylation reactions and act as the primary source for polysaccharide production. Uridine diphosphate (UDP)-glucose is one such valuable nucleotide sugar (retailing for around $1,000 per gram) applied in industry to synthesize unique pharmaceuticals and the development of new artificial sweeteners. Nucleotide sugars can be formed from a glycosyl monophosphate and a nucleotide monophosphate, as well as a monosaccharide synthon and a nucleotide diphosphate via chemical synthesis. However, enzymatic synthesis and metabolic engineering provide another route to produce nucleotide sugars by the application of recombinant proteins.

The purpose of this study is to develop a high-efficiency and inexpensive system to produce nucleotide sugars, especially UDP-glucose. Several strains of the yeast Pichia pastoris were constructed to generate nucleotide sugars from the glucose de novo biosynthesis pathway. In the process, three genes native to Pichia pastoris; hexokinase, phosphoglucomutase, and UDP-glucose pyrophosphorylase were introduced into the yeast genome and overexpressed under AOX1 high-yield inducible promoters. Optimal protein expression time was also measured and recorded. These enzymes enable increased production of UDP-glucose, providing a new potential method for nucleotide sugar production. We have also developed a generalized permeabilization method to the yeast cells to allow the UDP glucose produced inside the cells to be able to diffuse outwards into the extracellular environment. This would then allow for a more efficient method of collection for the product.