Production of Natural Deoxysugars for Chemical Synthesis of Glycosides

项目摘要 这里提出的建议的最终目标是生产自然产生的6-脱氧糖， 包括二脱氧糖、三脱氧糖、氨基糖和支链糖。这些特化的六角糖是 在植物和微生物的次生代谢物中被发现为重要的结构成分，经常发挥作用 在生物活性天然产品中发挥关键作用，如抗生素和抗癌疗法。 他们对糖工程的努力越来越感兴趣，这些努力的目的是改变在糖工程中发现的糖取代基 糖基化天然产品或在非糖基化天然产品中添加糖部分。 在自然界中，6-脱氧糖经常作为活化的dTDP-核苷的一部分被构建和修饰。 糖而不是独立的分子。我们建议研究一种新的方法来生产这些稀有的 脱氧糖使用我们最初开发的专利大肠杆菌系统来生产活性TDP-脱氧糖。至 要做到这一点，有许多挑战需要克服。首先，TDP-脱氧糖的效价需要 要增加，优先在生长介质中积累更多的物质。第二，有效的方式 需要从生长介质中分离和纯化分子。最后，脱氧糖 需要被水解和分离。 具体地说，在第一阶段，我们将论证使用D-岩藻糖生产脱氧糖的可行性 以dTDP-D-岩藻糖为模型衍生而来。这将通过1)增加dTDP-D-的产量来实现。 通过提高用于从TKDG生产dTDP-D-岩藻糖的酶Fcf1的表达和可溶性， 2)确定能够产生更大量dTDP-D-岩藻糖的化学定义的介质 介质，3)从化学定义的介质中开发dTDP-D-岩藻糖纯化方案，4)开发 一种dTDP-D-岩藻糖的水解和随后的D-岩藻糖的纯化方法。 在第二阶段，我们将扩大工艺，从dTDP生产至少18种额外的脱氧糖- 脱氧糖。将优化dTDP-脱氧糖的生产途径，以提高 在培养液中的含量至少达到500毫克/升，这将使我们能够从 单罐10个L发酵罐。在第三阶段，我们将单独和作为试剂盒将脱氧糖商业化，以 学术界和工业界的研究人员。通过扩大生产，第一阶段和第二阶段的工作也将 帮助支持我们的dTDP激活脱氧糖的销售。

Project Summary The ultimate goal of the proposal presented herein is to produce naturally occurring 6-deoxysugars, including di- and tri-deoxysugars, amino sugars and branched-chain sugars. These specialized hexoses are found as important structural components throughout plant and microbial secondary metabolites, often playing a crucial role in conferring activity in bioactive natural products, such as antibiotics and anticancer therapeutics. They are of increasing interest in glycoengineering efforts, which aim to alter sugar substituents found on glycosylated natural products or append sugar moieties to non-glycosylated natural products. In nature, 6-deoxysugars are frequently built up and modified as part of activated dTDP-nucleoside sugars instead of independent molecules. We propose to investigate a novel approach to produce these rare deoxysugars using a proprietary E. coli system we first developed to produce activated TDP-deoxysugars. To do this, there are a number of challenges which need to be overcome. First, titers of the TDP-deoxysugars need to be increased, preferentially accumulating more material in the growth medium. Second, an effective way to separate and purify the molecules from the growth medium needs to be developed. Finally, the deoxysugars need to be hydrolyzed and isolated. Specifically, in Phase I we will demonstrate the feasibility of deoxysugar production using D-fucose derived from dTDP-D-fucose as a model. This will be accomplished by 1) Increasing production of dTDP-D- fucose by enhancing expression and solubility of Fcf1, the enzyme used to make dTDP-D-fucose from TKDG, 2) determining a chemically defined medium capable of producing larger quantities of dTDP-D-fucose in the medium, 3) developing a dTDP-D-fucose purification protocol from the chemically defined medium, 4) developing a method for hydrolysis of dTDP-D-fucose and the subsequent purification of D-fucose. In Phase II, we will expand the process to manufacture at least 18 additional deoxysugars from dTDP- deoxysugars. Optimization of the dTDP-deoxysugar production pathways will be conducted to increase the amount in the medium to at least 500 mg/L. This will allow us to produce up to a gram of each deoxysugar from a single 10 L fermenter. In Phase III, we will commercialize the deoxysugars, individually and as a kit, to researchers in academia and industry. Through expanded production, the work done in Phases I and II will also help support the sale of our dTDP-activated deoxysugars.