Production of Natural Deoxysugars for Use in Chemical Synthesis of Glycosides

项目摘要 本文提出的建议的目标是生产天然存在的6-脱氧糖，包括二-脱氧糖。 和三脱氧糖、氨基糖和支链糖。这些特殊的己糖被发现为 重要的结构成分贯穿植物和微生物的次级代谢产物，通常起着至关重要的作用。 在赋予生物活性天然产物如抗生素和抗癌治疗剂活性中的作用。他们是 在糖工程的努力，其目的是改变糖基化上发现的糖取代基， 天然产物或将糖部分附加到非糖基化天然产物。 在自然界中，6-脱氧糖经常被构建和修饰为活化的dTDP-核苷的一部分， 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罕见的 脱氧糖使用专有的E.我们首先开发的大肠杆菌系统生产活化的TDP-脱氧糖。到 要做到这一点，在第一阶段可行性研究中有几个挑战需要解决。首先，TDP的滴度- 需要增加脱氧糖的含量，优先在生长培养基中积累更多的物质。第二、 需要开发一种从生长培养基中分离和纯化分子的有效方法。最后， 需要水解和分离脱氧糖。 在阶段I中，我们证明了使用衍生自dTDP的D-岩藻糖生产脱氧糖的可行性。 D-岩藻糖作为模型。这是通过以下方式实现的：1）通过增强dTDP-D-岩藻糖的产量来增加dTDP-D-岩藻糖的产量。 Fcf 1的表达和溶解度，所述酶用于从TKDG制备dTDP-D-岩藻糖，2）确定 能够在培养基中产生更大量的dTDP-D-岩藻糖的培养基，3）产生dTDP-D-岩藻糖 4）开发用于水解dTDP-D-岩藻糖的方法， 随后纯化D-岩藻糖。 在第二阶段，我们将扩大该工艺，从dTDP生产18种额外的脱氧糖- 脱氧糖将进一步优化dTDP-脱氧糖生产途径，以增加 在培养基中的添加量为0.5- 1.0g/L。这将使我们能够从一种脱氧糖中生产高达2.5克的每种脱氧糖。 单个10 L发酵罐。在第三阶段，我们将商业化脱氧糖，单独和作为一个试剂盒， 学术界和工业界的研究人员。通过扩大生产，第二阶段的工作也将有助于 增加dTDP活化的脱氧糖作为重要的次级产物的产量。

Project Summary The 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 were several challenges to address in the Phase I Feasibility study. First, titers of the TDP- deoxysugars needed 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 needed to be developed. Finally, the deoxysugars needed to be hydrolyzed and isolated. In Phase I we demonstrated the feasibility of deoxysugar production using D-fucose derived from dTDP- D-fucose as a model. This was 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 medium capable of producing larger quantities of dTDP-D-fucose in the medium, 3) developing a dTDP-D-fucose purification protocol from the 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 18 additional deoxysugars from dTDP- deoxysugars. Further optimization of the dTDP-deoxysugar production pathways will be conducted to increase the amount in the medium to 0.5-1.0 g/L. This will allow us to produce up to a 2.5 g 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 Phase II will also help increase production of the dTDP-activated deoxysugars as important secondary products.