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

高产生产 GDP-岩藻糖用于分子岩藻糖基化

• 批准号: 9040999
• 负责人: Leila Aminova
• 金额: $ 56.64万
• 依托单位: ZUCHEM, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9040999
• 关键词: Anti-Infective Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antigens; Antineoplastic Agents; Bacteria; Biochemical; Biological Process; Blood; Blood Group Antigens; Cell physiology; Cells; Communities; Custom; Development; Engineering; Enzymes; Escherichia coli; Fermentation; Formulation; Fucose; Fucosyltransferase; Genes; Genetic Engineering; Glucose; Goals; Guanosine Diphosphate Fucose; Guanosine Diphosphate Mannose; Guanosine Triphosphate; Health; Human; Human Milk; In Situ; Infection; Licensing; Link; Lipids; Marketing; Methodology; Methods; Modification; Neoplasm Metastasis; Nutrient; Nutritional; Oligosaccharides; Pharmacologic Substance; Phase; Pichia; Play; Polysaccharides; Price; Process; Production; Productivity; Property; Proteins; Reaction; Research; Role; Saccharomyces cerevisiae; Source; System; Technology; Testing; Toxin; Uridine Diphosphate Sugars; Vaccines; Yeasts; anti-cancer therapeutic; antimicrobial; base; cancer cell; cofactor; commercialization; cost; drug discovery; extracellular; glycosyltransferase; in vivo; interest; large scale production; meetings; metabolic engineering; microbial; milligram; novel; novel therapeutics; nucleoside triphosphate; overexpression; promoter; small molecule; success; sugar; sugar nucleotide

 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 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.

 描述（由申请人提供）：岩藻糖基化在许多细胞过程中起重要作用。细胞中的岩藻糖基化寡糖参与许多生化识别过程、微生物感染、毒素进入和癌细胞转移。这些性质使得岩藻糖基化分子对于药物和药物发现的需要是有价值的，但是目前的生产方法非常昂贵并且不切实际。最值得注意的是生产活性糖GDP-岩藻糖的费用和困难。我们的目标是增加GDP-岩藻糖和岩藻糖基化分子（如寡糖）的可及性，以便研究界能够更好地了解这些化合物在人类健康中的作用，开发新型抗菌、抗炎和抗癌药物，并开发适合大规模生产各种寡糖和岩藻糖基化分子的菌株。 迄今为止描述的使用化学酶促合成或修饰的E. coli和革兰氏阳性菌S.酿酒酵母菌株都只产生少量的物质或 过于复杂，无法扩展。在此，我们建议开发一种全新的基于酵母的生产GDP-岩藻糖的方法。这种基于酵母的系统有两个主要优点。首先，在葡萄糖存在下，它使用诱导型启动子过量表达两种能够将天然丰富来源的GDP-甘露糖转化为GDP-岩藻糖的酶。其次，它利用核苷酸-糖转运蛋白进行GDP-岩藻糖的细胞外释放。该系统还允许使用额外的酶用于体内合成靶分子的可能性。 在阶段I中，我们通过开发能够以高产率产生GDP-甘露糖的酵母菌株、过量表达将GDP-甘露糖转化为GDP-岩藻糖所需的酶并证明以高产率产生GDP-岩藻糖的能力，证明了使用该方法的可行性。我们还证明了将GDP-岩藻糖转运出细胞的能力，并确定了发酵的初始条件。在第II阶段，我们将进一步设计和优化GDP-岩藻糖的生产，并通过检测几种重要的岩藻糖基化分子（如人乳寡糖和岩藻糖基化蛋白质）的生产来证明其实用性，这些分子的生产来自我们容易获得的起始材料。最后，III期商业化将涉及销售GDP-岩藻糖，许可该系统用于各种应用，并使用该系统生产定制的岩藻糖基化寡糖、小分子和蛋白质。