High-yield production of GDP-fucose for fucosylation of molecules

DESCRIPTION provided by applicant Fucosylation plays an important role in many cellular processes Fucosylated oligosaccharides in the cell are involved in many biochemical recognition processes microbial infections toxin entry and cancer cell metastasis These properties make fucosylated molecules valuable for pharmaceutical and drug discovery needs but current production methods are very expensive and impractical Most notably is the expense and difficulty in producing the activated sugar GDP fucose Our goal is to increase accessibility of GDP fucose and fucosylated molecules such as oligosaccharides so that the research community can better understand the role of these compounds in human health develop novel antimicrobial anti inflammatory and anti cancer agents and develop strains suitable for large scale production of various oligosaccharides and fucosylated molecules Current methods described to date for the production of GDP fucose using either chemoenzymatic synthesis or modified E coli and S cerevisiae strains all yield only small milligram quantities of material or are overly complicated and canandapos t be scaled Here we propose to develop an entirely new yeast based method for production of GDP fucose There are two main advantages to this this yeast based system First it uses an inducible promoter in the presence of glucose to overexpress two enzymes capable of converting a naturally abundant source of GDP mannose to GDP fucose Second it utilizes a nucleotide sugar transporter for the extracellular release of GDP Fucose The system also allows the possibility of using additional enzymes for in vivo synthesis of target molecules In Phase I we demonstrated the feasibility of using this approach by developing a yeast strain that can produce GDP mannose at high yields overexpressing the enzymes necessary to convert GDP mannose to GDP fucose and demonstrating the ability to produce GDP fucose at high yields We have also demonstrated the ability to transport GDP fucose out of the cell and have determined the initial conditions for fermentation In Phase II we will further engineer and optimize the production of GDP fucose and demonstrate its utility by testing the production of several important fucosylated molecules such as human milk oligosaccharides and fucosylated proteins from starting materials that are readily available to us Finally Phase III commercialization will involve selling GDP fucose licensing the system for use in a variety of applications and using the system to produce custom fucosylated oligosaccharides small molecules and proteins PUBLIC HEALTH RELEVANCE We propose to develop a yeast based system for the production of GDP fucose in vivo at large scale This technology will make this sugar nucleotide economically available for modification of natural sugars lipids proteins antibiotics antibodie and vaccines The proposed research has the potential to open up several multi billion dollar markets in anti infective and anticancer therapeutics as well as having application in the production of nutritional oligosaccharides