Production of Natural Deoxysugars for Use in Chemical Synthesis of Glycosides

用于糖苷化学合成的天然脱氧糖的生产

• 批准号: 10822937
• 负责人: Robert J. Turner
• 金额: $ 93.02万
• 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10822937
• 关键词: Academia; Acids; Address; Amino Sugars; Antibiotics; Antineoplastic Agents; Bacterial Antibiotic Resistance; Biological; Cells; Deoxy Sugars; Derivation procedure; Development; Enhancers; Enzymes; Escherichia coli; Feasibility Studies; Formulation; Fucose; Genes; Glucose; Glycoengineering; Glycosides; Goals; Growth; Hexoses; Hydrolysis; Individual; Industry; Methods; Midwestern United States; Modeling; Modification; Natural Products; Nature; Nucleosides; Pathway interactions; Phase; Plants; Plasmids; Play; Preparation; Process; Production; Property; Protocols documentation; Research Personnel; Role; Sales; Solubility; System; Therapeutic; Work; anti-cancer therapeutic; bioactive natural products; bioprocess; chemical synthesis; commercialization; glycosylation; improved; in vivo; innovation; interest; maltose-binding protein; manufacture; microbial; natural product derivative; novel; novel strategies; novel therapeutics; research and development; secondary metabolite; sugar

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.

项目概要 本文提出的提案的目标是生产天然存在的 6-脱氧糖，包括二- 以及三脱氧糖、氨基糖和支链糖。这些特殊的己糖被发现为 整个植物和微生物次生代谢产物的重要结构成分，通常发挥着至关重要的作用 在赋予生物活性天然产物（例如抗生素和抗癌疗法）活性方面发挥作用。他们是 人们对糖工程努力越来越感兴趣，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的天然产物上。 在自然界中，6-脱氧糖经常作为活化的 dT​​DP-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方法来生产这些稀有的 脱氧糖使用我们首先开发的专有大肠杆菌系统来生产活化的 TDP-脱氧糖。到 为此，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，优先在生长培养基中积累更多物质。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， 脱氧糖需要水解和分离。 在第一阶段，我们证明了使用源自 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 Fcf1 的表达和溶解度，Fcf1 是用于从 TKDG 制备 dTDP-D-岩藻糖的酶，2) 确定 能够在培养基中产生大量 dTDP-D-岩藻糖的培养基，3) 开发 dTDP-D-岩藻糖 培养基的纯化方案，4) 开发一种水解 dTDP-D-岩藻糖的方法和 随后纯化D-岩藻糖。 在第二阶段，我们将扩大工艺，从 dTDP- 中生产另外 18 种脱氧糖。 脱氧糖。将进一步优化 dTDP-脱氧糖生产途径，以提高 培养基中的量为0.5-1.0g/L。这将使我们能够从脱氧糖中生产最多 2.5 克的脱氧糖。 单个 10 L 发酵罐。在第三阶段，我们将单独和作为套件将脱氧糖商业化，以 学术界和工业界的研究人员。通过扩大生产，二期所做的工作也将有助于 增加 dTDP 激活的脱氧糖作为重要副产品的产量。