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发挥着重要作用。细胞中的岩藻糖基化寡糖参与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制药和药物发现需求有价值，但目前的生产方法生产非常昂贵且不切实际。最值得注意的是生产活性糖 GDP-岩藻糖的费用和难度。迄今为止描述的使用化学酶合成或修饰微生物（例如大肠杆菌和酿酒酵母）生产 GDP-岩藻糖的方法要么产量太少，要么过于复杂且无法规模化。在这里，我们建议开发一种基于使用酵母系统生产 GDP-岩藻糖的全新系统。该系统使用强诱导型启动子在甲基营养酵母中从头开始途径过度表达酶。这些酵母自然产生高产量的 GDP-甘露糖，所提出的系统将其转化为 GDP-岩藻糖，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点是高水平表达涉及 GDP-岩藻糖合成和运输的蛋白质，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能够以最高产量生产 GDP-甘露糖的酵母，过表达将 GDP-甘露糖转化为 GDP-岩藻糖所需的酶，并测试生产 GDP-L-岩藻糖的能力。然后我们将测试将 GDP-L-岩藻糖转运出细胞的能力并确定发酵的初始条件。在第二阶段，我们将进一步设计和优化 GDP-岩藻糖的生产，并通过测试使用我们现成的原材料生产几种母乳来证明其效用。最后，第三阶段商业化将涉及销售 GDP-岩藻糖、授权该系统供多家公司使用，以及使用它来生产定制岩藻糖基化寡糖、小分子和蛋白质。