Rapid Multiplexed Nanoprobe Assays for Pluripotent Stem Cell Differentiation

DESCRIPTION: Fucosylation plays an important role in much cellular process. Fucosylated oligosaccharides in the cell are involved in various types of biochemical recognition processes and in microbial infections, toxin entry, and cancer cells metastasis.These properties make these carbohydrates valuable for pharmaceutical and drug discovery needs but current production methods production is very expensive and impractical. Most notably is the expense and difficulty in producing the activated sugar, GDP-fucose. Current methods described to date for the production of GDP-fucose using chemoenzymatic synthesis, or modified microorganisms such E. coli and S. cerevisiae all either yield too small quantities of material or are overly complicated and can't be scaled. Here we propose to develop an entirely new system based on the use of a yeast system for production GDP-fucose. This system uses a strong inducible promoter for overexpression of the enzyme from a de novo pathway in methylotrophic yeast. These yeast naturally produce a high yield of GDP-mannose which the proposed system will convert to GDP-fucose and utilize nucleotide-sugar transporter for extracellular release. We foresee the advantages of this approach to be high- level expression of protein involved to GDP-fucose synthesis and transport and the possibility to use the desired enzymes for in vivo synthesis. In Phase I we will test the feasibility of developing this system by screening a set of yeast that can produce GDP-mannose in highest yield, overexpress the enzymes necessary to convert GDP-mannose to GDP-fucose, and test the ability to produce GDP-L-fucose. We will then test the ability to transport GDP-L- fucose out of the cell and determine the initial conditios for fermentation. In Phase II wewill further engineer and optimize the production of GDP-fucose and demonstrate its utility by testing the production of several human milk from starting materials that are readily available to us. Finally, Phase III commercialization will involve sellngGDP-fucose, licensing the system for use by a variety of companies, and in using it to produce custom fucosylated oligosaccharides, small molecules, and proteins. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Development of the yeast gene expression system proposed here will allow production of GDP-fucose in vivo at a large scale. This technology will make this sugar nucleotide available in low price for modification of already synthesized or natural sugar, lipids, proteins, antibiotics andvaccines. The proposed research has the potential to open up several multi-billion dollar markets in anti-infective and anticancer therapeutics.