Production of Activated TDP-Deoxysugars in E. coli

描述(申请人提供)：本提案的最终目标是利用大肠杆菌作为全细胞生物催化剂来生产多种TDP-脱氧糖，包括二和三脱氧糖、氨基糖和支链糖。这些专门化的活性己糖是植物和微生物次生代谢物中的重要结构成分，在抗生素和抗癌药物等生物活性天然产物中起着至关重要的作用。在第一阶段，我们提出了一种通过代谢工程在大肠杆菌中生产稀有TDP-脱氧糖的新方法。在第一阶段的研究中，我们成功地修饰了大肠杆菌AB707，通过灭活和过度表达特定途径的酶来积累TDP-4-酮-6-脱氧-D-葡萄糖(TKDG；TDP-脱氧糖的关键中间体)。利用外源TDP-脱氧糖生物合成酶，积累的TKDG池被转化为特定的TDP-脱氧糖，产量高达80 mg/L，超过了我们的第一阶段目标。可行性研究的成功将在第二阶段扩大。具体地说，我们将通过额外的基因工程实验进一步提高TDP-脱氧糖的产量，以限制葡萄糖-磷酸盐在竞争途径中的损失，增加胸苷三磷酸(TTP)池，减少葡萄糖对分解代谢的抑制，并优化发酵条件。我们还将通过克隆和表达几个外源糖生物合成基因来扩大我们的TDP-脱氧糖产品的范围，以生产0个额外的复杂TDP-脱氧糖。在第三阶段，我们将通过与战略合作伙伴开展合作研发计划来制造新的糖结合物，通过提供TDP-脱氧糖以及作为研究试剂的脱氧糖，以及通过在特定应用的基础上授权该技术，将该技术商业化。

DESCRIPTION (provided by applicant): The ultimate goal of the proposal presented herein is to use E. coli as whole cell biocatalysts for the production of a wide variety of TDP-deoxysugars including di- and tri-deoxysugars, amino sugars and branched-chain sugars. These specialized activated 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. In Phase I, we proposed to investigate a novel approach to produce rare TDP-deoxysugars in Escherichia coli through metabolic engineering. During the Phase I study we successfully modified E. coli AB707 to accumulate TDP-4-keto-6-deoxy-D-glucose (TKDG; a key intermediate of TDP-deoxysugars) through inactivations and overexpression of specific pathway enzymes. Using exogenous TDP-deoxysugar biosynthetic enzymes, the accumulated TKDG pools were converted to specific TDP-deoxysugars in yields up to 80 mg/L, exceeding our Phase I goal. The success of the feasibility study will be expanded in Phase II. Specifically, we will further increase TDP- deoxysugar yields through additional genetic engineering experiments to limit loss of glucose- -phosphate to competing pathways, increase thymidine triphosphates (TTP) pools, reduce catabolite repression by glucose, and optimize fermentation conditions. We will also expand our range of TDP-deoxysugar products by cloning and expressing several exogenous sugar biosynthetic genes for the production of 0 additional complex TDP-deoxysugars. In Phase III we will commercialize the technology by carrying out Partnered R&D programs with strategic partners to make new glycoconjugates, by offering TDP-deoxysugars as well as deoxysugars as research reagents, and by licensing the technology on an application-specific basis.