Production of Natural Deoxysugars for Chemical Synthesis of Glycosides

化学合成糖苷用天然脱氧糖的生产

• 批准号: 10384863
• 负责人: Robert J. Turner
• 金额: $ 21.74万
• 依托单位: MIDWEST BIOPROCESSING CENTER, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-17 至 2023-03-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384863
• 关键词: Academia; Acids; Address; Amino Sugars; Antibiotics; Antineoplastic Agents; Bacterial Antibiotic Resistance; Biological; Cells; Chemicals; Complex; Deoxy Sugars; Development; Enzymes; Escherichia coli; Fermentation; Fucose; Glucose; Glycoengineering; Glycosides; Goals; Growth; Hexoses; Hydrolysis; Individual; Industry; Methods; Midwestern United States; Modeling; Natural Products; Nature; Nucleosides; Pathway interactions; Phase; Plants; Play; Preparation; Process; Production; Property; Protocols documentation; Research Personnel; Role; Running; Sales; Solubility; System; Testing; Therapeutic; Time; Work; anti-cancer therapeutic; antineoplastic antibiotics; bioactive natural products; bioprocess; chemical synthesis; design; improved; in vivo; innovation; interest; maltose-binding protein; microbial; novel; novel strategies; novel therapeutics; research and development; sugar

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.

项目摘要 本文提出的提案的最终目标是生产自然发生的6-脱氧糖果， 包括Di-di-deoxysugars，氨基糖和分支链糖。这些专业的己糖是 在整个植物和微生物二级代谢物中发现了重要的结构成分，经常播放 在赋予生物活性天然产物（例如抗生素和抗癌疗法）中的活性中至关重要的作用。 他们对糖工程的兴趣越来越兴趣，旨在改变糖取代基 糖基化天然产物或将糖部分添加到非糖基化天然产物中。 在自然界中，6-脱氧糖果经常作为激活的DTDP-核苷的一部分建立和修改 糖代替独立分子。我们建议研究一种新的方法来产生这些罕见的方法 使用专有的大肠杆菌系统的脱氧剂，我们首先开发出来生产活化的TDP-脱氧糖。到 这样做，需要克服许多挑战。首先，TDP-Deoxysugars的滴度需要 要增加，优先在生长培养基中积累了更多材料。第二，一种有效的方法 需要开发与生长培养基的分子分离并净化分子。最后，Deoxysugars 需要水解和隔离。 具体而言，在第一阶段，我们将使用D-纤维糖来证明脱氧果糖生产的可行性 源自DTDP-D-凝血作为模型。这将通过1）增加DTDP-D-的产生 通过增强FCF1的表达和溶解度，该酶用于从TKDG制造DTDP-D-ucose的酶， 2）确定能够在该化学定义的培养基 介质，3）从化学定义的培养基中开发DTDP-DP-D-凝血纯化方案，4） DTDP-D-葡萄糖水解的方法和随后的D-凝糖纯化。 在第二阶段，我们将扩大从DTDP-至少生产18个其他脱氧剂的过程 Deoxysugars。将进行DTDP-脱氧果糖生产途径的优化以增加 在培养基中至少为500 mg/l。这将使我们从中产生多达每克的每克 一个10 L发酵罐。在第三阶段，我们将单独和作为套件将脱氧糖果商业化为 学术界和工业的研究人员。通过扩大的生产，第I和II阶段完成的工作也将 帮助支持我们DTDP激活的Deoxysugars的销售。