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

 描述(申请人提供)：岩藻糖基化在许多细胞过程中起着重要作用。细胞中的岩藻糖化低聚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。这些性质使得岩藻糖化分子在制药和药物开发需求中具有很高的价值，但目前的生产方法非常昂贵和不切实际。最值得注意的是生产活性糖GDP-岩藻糖的费用和难度。我们的目标是增加GDP-岩藻糖和岩藻糖化分子(如低聚糖)的可及性，以便研究界能够更好地了解这些化合物对人类健康的作用，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低聚糖和岩藻糖分子的菌株。目前所描述的生产GDP-岩藻糖的方法要么是使用化学酶合成，要么是使用改良的大肠杆菌和酿酒酵母菌株，这些方法都只能产生少量的物质或 都过于复杂，而且无法扩展。在这里，我们建议开发一种全新的以酵母为基础的生产GDP-岩藻糖的方法。这种以酵母为基础的系统有两个主要优点。首先，它使用一个可诱导的启动子，在葡萄糖存在的情况下，过度表达两种酶，能够将天然丰富的GDP-甘露糖转化为GDP-岩藻糖。其次，它利用一种核苷酸-糖转运体在细胞外释放GDP-岩藻糖。该系统还允许使用额外的酶在体内合成目标分子。在第一阶段，我们通过开发一株能够高产生产GDP-甘露糖的酵母菌株，过表达将GDP-甘露糖转化为GDP-岩藻糖所需的酶，并展示了以高产量生产GDP-岩藻糖的能力，证明了使用这种方法的可行性。我们还展示了将GDP-岩藻糖运输出细胞的能力，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GDP-岩藻糖的生产，并通过测试几个重要的岩藻糖化分子(如人乳低聚糖和岩藻糖化蛋白)的生产来展示其效用，这些分子是从我们容易获得的原料中生产出来的。最后，第三阶段的商业化将包括销售GDP-岩藻糖，授权该系统用于各种应用，并使用该系统生产定制的岩藻糖化低聚糖、小分子和蛋白质。

 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.