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

基本信息

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
项目类别：
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

项目摘要

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）：岩藻糖基化在细胞中的许多细胞过程中发挥重要作用，涉及许多生化识别过程、微生物感染、毒素进入和癌细胞转移，这些特性使得岩藻糖基化分子对于药物和癌症具有重要价值。药物发现需要，但目前的生产方法非常昂贵且不切实际，最值得注意的是生产活性糖 GDP-岩藻糖的成本和难度。 GDP-岩藻糖和岩藻糖基化分子如寡糖，使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药物的菌株迄今为止描述的使用化学酶合成或改良的大肠杆菌和酿酒酵母菌株生产GDP-岩藻糖的方法均仅产生少量毫克。材料数量过于复杂且无法规模化。在此，我们建议开发一种全新的基于酵母的方法来生产 GDP-岩藻糖。这种基于酵母的系统有两个主要优点，首先，它使用诱导型启动子。其次，它利用核苷酸-糖转运蛋白将 GDP-岩藻糖释放到细胞外。使用额外的酶体内合成目标分子的可能性在第一阶段，我们通过开发一种可以高产量生产 GDP-甘露糖的酵母菌株，过度表达将 GDP-甘露糖转化为所需的酶，证明了使用这种方法的可行性。 GDP-岩藻糖并展示了高产量生产 GDP-岩藻糖的能力我们还展示了将 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二阶段，我们将进一步设计和优化发酵。生产GDP-岩藻糖，并通过测试几种重要的岩藻糖基化分子（例如母乳寡糖和岩藻糖基化蛋白质）的生产来证明其效用，这些分子是我们容易获得的起始材料。最后，第三阶段商业化将涉及销售 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。