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：
8592879
负责人：
Leila Aminova
金额：
$ 18.7万
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 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8592879
关键词：
Anabolism Anti-Infective Agents Antibiotics Bacteria Biochemical Biological Process Carbohydrates Cell physiology Cells Contracts Culture Media Custom Development Engineering Enzymes Escherichia coli Fermentation Fucose Fucosyltransferase Gene Expression Genetic Engineering Glucosephosphate Dehydrogenase Goals Growth Guanosine Diphosphate Fucose Guanosine Diphosphate Mannose Guanosine Triphosphate Human Human Milk Hydro-Lyases In Situ Infection Licensing Link Lipids Marketing Methods Modification NADP Neoplasm Metastasis Nutritional Oligosaccharides Oxidoreductase Pathway interactions Pharmacologic Substance Phase Play Polysaccharides Price Process Production Property Proteins Reaction Recycling Research Rhamnose Role Saccharomyces cerevisiae System Technology Testing Toxin Uridine Diphosphate Sugars Vaccines Yeasts anti-cancer therapeutic base cancer cell commercialization cost drug discovery epimerase extracellular glycosyltransferase improved in vivo large scale production meetings microbial microorganism novel novel therapeutics nucleoside triphosphate overexpression promoter protein expression public health relevance screening small molecule success sugar sugar nucleotide

项目摘要

DESCRIPTION: 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.

描述：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了各种类型的生化识别过程以及微生物感染，毒素进入和癌细胞转移。这些特性使这些碳水化合物对于药物和药物发现需求很有价值，但是当前的生产方法的生产非常昂贵且不切实际。最值得注意的是产生活化糖，GDP-诱饵的费用和困难。迄今为止，使用化学酶合成或改性的微生物（例如大肠杆菌和酿酒酵母）生产GDP凝糖的当前方法，所有的方法要么产生太少的材料，要么过于复杂并且无法缩放。在这里，我们建议基于使用酵母系统进行生产GDP企业开发一个全新的系统。该系统使用强大的诱导启动子来过表达甲基营养酵母中从头途径的酶。这些酵母自然会产生高产量的GDP甘露糖，提出的系统将转换为GDP-葡萄糖并利用核苷酸 - 糖转运蛋白进行细胞外释放。我们预见，这种方法的优势是与GDP凝集合成和运输有关的蛋白质的高水平表达，以及使用所需酶进行体内合成的可能性。在第一阶段，我们将通过筛选一组来测试开发该系统的可行性可以以最高产量产生GDP甘露糖的酵母，过表达将GDP-甘露糖转换为GDP-观糖所需的酶，并测试产生GDP-L-L-观糖的能力。然后，我们将测试将GDP-L-富糖从细胞中运出的能力，并确定发酵的初始孔。在第二阶段，我们将进一步设计并优化GDP葡萄糖的生产，并通过测试从起始材料中测试几种人牛奶的生产，从而证明其实用性。最后，第三阶段的商业化将涉及卖出GDP-ucose，许可各种公司使用的系统，并在使用它生产自定义的岩藻糖基化寡糖，小分子和蛋白质时。