Production of Natural Deoxysugars for Chemical Synthesis of Glycosides

项目摘要 本文提出的建议的最终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。这些特殊的己糖是 在整个植物和微生物的次生代谢产物中发现重要的结构成分，经常发挥作用， 在赋予生物活性天然产物如抗生素和抗癌治疗剂活性方面起着至关重要的作用。 他们在糖工程努力中越来越感兴趣，其目的是改变在糖上发现的糖取代基。 糖基化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，需要克服一些挑战。首先，TDP-脱氧糖的滴度需要 增加，优先在生长培养基中积累更多的材料。第二，一个有效的方法， 从生长培养基中分离和纯化分子的技术需要发展。最后，脱氧糖 需要水解和分离。 具体来说，在第一阶段，我们将证明使用D-岩藻糖生产脱氧糖的可行性 衍生自dTDP-D-岩藻糖作为模型。这将通过以下方式实现：1）增加dTDP-D的产量， 通过增强Fcf 1的表达和溶解度来增加岩藻糖，Fcf 1是用于从TKDG制备dTDP-D-岩藻糖的酶， 2)确定化学成分确定的培养基，所述化学成分确定的培养基能够在所述培养基中产生更大量的dTDP-D-岩藻糖， 培养基，3）开发从化学成分确定的培养基中纯化dTDP-D-岩藻糖的方案，4）开发 一种水解dTDP-D-岩藻糖并随后纯化D-岩藻糖的方法。 在第二阶段，我们将扩大该工艺，从dTDP中生产至少18种额外的脱氧糖。 脱氧糖将进行dTDP-脱氧糖生产途径的优化，以增加 在培养基中的量为至少500 mg/L。这将使我们能够生产每克脱氧糖， 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，第一阶段和第二阶段的工作还将 帮助支持我们的dTDP活化脱氧糖的销售。

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.