SBIR-STTR Award

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 we will 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 sellng GDP-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 Statement:


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 and vaccines. The proposed research has the potential to open up several multi-billion dollar markets in anti-infective and anticancer therapeutics.

Project Terms:
Anabolism; anti-cancer therapeutic; Anti-Infective Agents; Antibiotics; Bacteria; base; Biochemical; Biological Process; cancer cell; Carbohydrates; Cell physiology; Cells; commercialization; Contracts; cost; Culture Media; Custom; Development; drug discovery; Engineering; Enzymes; epimerase; Escherichia coli; extracellular; Fermentation; Fucose; Fucosyltransferase; Gene Expression; Genetic Engineering; Glucosephosphate Dehydrogenase; glycosyltransferase; Goals; Growth; Guanosine Diphosphate Fucose; Guanosine Diphosphate Mannose; Guanosine Triphosphate; Human; Human Milk; Hydro-Lyases; improved; In Situ; in vivo; Infection; large scale production; Licensing; Link; Lipids; Marketing; meetings; Methods; microbial; microorganism; Modification; NADP; Neoplasm Metastasis; novel; novel therapeutics; nucleoside triphosphate; Nutritional; Oligosaccharides; overexpression; Oxidoreductase; Pathway interactions; Pharmacologic Substance; Phase; Play; Polysaccharides; Price; Process; Production; Promotor (Genetics); Property; protein expression; Proteins; public health relevance; Reaction; Recycling; Research; Rhamnose; Role; Saccharomyces cerevisiae; screening; small molecule; success; sugar; sugar nucleotide; System; Technology; Testing; Toxin; Uridine Diphosphate Sugars; Vaccines; Yeasts

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 can'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 Statement:


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.

Project Terms:
anti-cancer therapeutic; Anti-Infective Agents; Anti-inflammatory; Anti-Inflammatory Agents; Antibiotics; Antigens; antimicrobial; Antineoplastic Agents; Bacteria; base; Biochemical; Biological Process; Blood; Blood Group Antigens; cancer cell; Cell physiology; Cells; cofactor; commercialization; Communities; cost; Custom; Development; drug discovery; Drug Formulations; Engineering; Enzymes; Escherichia coli; extracellular; Fermentation; Fucose; Fucosyltransferase; Genes; Genetic Engineering; Glucose; glycosyltransferase; Goals; Guanosine Diphosphate Fucose; Guanosine Diphosphate Mannose; Guanosine Triphosphate; Health; Human; Human Milk; In Situ; in vivo; Infection; interest; large scale production; Licensing; Link; Lipids; Marketing; meetings; metabolic engineering; Methodology; Methods; microbial; milligram; Modification; Neoplasm Metastasis; novel; novel therapeutics; nucleoside triphosphate; Nutrient; Nutritional; Oligosaccharides; overexpression; Pharmacologic Substance; Phase; Pichia; Play; Polysaccharides; Price; Process; Production; Productivity; Promotor (Genetics); Property; Proteins; public health relevance; Reaction; Research; Role; Saccharomyces cerevisiae; small molecule; Source; success; sugar; sugar nucleotide; System; Technology; Testing; Toxin; Uridine Diphosphate Sugars; Vaccines; Yeasts