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

聚焦化在许多细胞过程中起重要作用。细胞中聚集的寡糖参与多种类型的生化识别过程，参与微生物感染、毒素进入和癌细胞转移。这些特性使这些碳水化合物对制药和药物发现有价值，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的成本和难度，以GDP为重点。目前所描述的生产GDP的方法——主要是利用化学酶合成，或修饰的微生物，如大肠杆菌和酿酒杆菌，要么产量过少，要么过于复杂，无法规模化。在这里，我们建议开发一种基于酵母系统的全新系统，以生产GDP为重点。该系统使用一个强诱导启动子从甲基营养酵母的从头途径过表达酶。这些酵母自然产生高产量的GDP-甘露糖，该系统将其转化为GDP-聚焦并利用核苷酸-糖转运蛋白进行细胞外释放。我们预见到这种方法的优点是高水平表达参与GDP聚焦合成和运输的蛋白质，并有可能使用所需的酶进行体内合成。在第一阶段，我们将通过筛选一组

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.