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（由适用提供）：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的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。这些特性使葡萄糖基化的分子对于药物和药物发现需求有价值，但是当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化的糖GDP诱饵的费用和困难。我们的目标是提高GDP-葡萄糖和诱导分子（例如寡糖）的可访问性，以便研究界可以更好地了解这些化合物在人类健康中的作用，开发新型抗菌素，抗炎性和抗癌剂，并开发适合于各种寡糖糖的大量生产的菌株。迄今为止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-凝糖的当前方法仅产生少量毫克材料或过于复杂，无法缩放。在这里，我们建议开发一种全新的基于酵母的方法来生产GDP企业。该基于酵母的系统有两个主要优势。首先，在存在葡萄糖的情况下，它使用诱导启动子来过表达两种能够将自然丰富的GDP甘露糖来转化为GDP-浓度的酶。其次，它利用核苷酸 - 糖转运蛋白来细胞外释放GDP-Fucusose。该系统还允许使用其他酶进行靶分子的体内合成。在第一阶段，我们证明了通过开发可以以高产量产生GDP甘露糖的酵母菌菌株来使用这种方法的可行性，从而过表达将GDP甘露糖转化为GDP糖糖糖糖糖糖糖的酶，并证明了以高产量产生GDP型葡萄糖的能力。我们还证明了将GDP葡萄糖从细胞中运输出来的能力，并确定了发酵的初始条件。在第二阶段，我们将进一步设计并优化GDP凝血糖的产生，并通过测试几种重要的岩藻糖基化分子（例如人乳寡糖）和诱导型蛋白质的产生来证明其实用性，并从我们很容易获得的起始材料中使用。最后，第三阶段的商业化将涉及出售GDP-凝血蛋白，许可在各种应用中使用的系统，并使用该系统生产自定义的岩藻糖基化的寡糖，小分子和蛋白质。