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.

项目摘要 本文提出的提案的目的是生产自然发生的6-脱氧剂，包括di- 以及三氧合，氨基糖和分支链糖。这些专业的己糖被发现 整个植物和微生物二级代谢产物的重要结构成分，经常发挥至关重要的作用 在赋予生物活性天然产品（例如抗生素和抗癌疗法）中的活性中的作用。他们是 越来越多地兴趣糖化工作，旨在改变糖基化的糖取代基 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，在I期可行性研究中需要解决一些挑战。首先，TDP的滴度 需要增加Deoxysugars，优先在生长培养基中积累更多的材料。第二， 需要开发分子与生长培养基中分离和净化分子的有效方法。最后， 需要水解和分离脱氧。 在第一阶段，我们证明了使用dtdp-衍生的D-凝糖脱氧糖产生的可行性 D-凝血作为模型。这是通过1）通过增强DTDP-D-D-ucose的产生来实现的 FCF1的表达和溶解度，用于从TKDG制造DTDP-D-ucose的酶，2）确定 能够在培养基中产生大量DTDP-D-ucose的培养基，3）开发DTDP-D-D-ucose 培养基的纯化方案，4）开发一种用于DTDP-D-D-观糖的水解方法 随后纯化D-凝糖。 在第二阶段，我们将扩大该过程，从DTDP-生产18个额外的Deoxysugars Deoxysugars。将进一步优化DTDP-Deoxysugar生产途径以增加 培养基的量为0.5-1.0 g/l。这将使我们能够从一个 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果商业化为 学术界和工业的研究人员。通过扩大的生产，第二阶段完成的工作也将有助于 增加了DTDP激活的脱氧剂作为重要的二级产品的产生。