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。我们提出了一种在大肠杆菌中通过代谢工程生产稀有TDP-脱氧糖的新方法。通过结合分别导致葡萄糖-6-磷酸(G6P；TKDG的中间体)和TDP-4-酮-6-脱氧-D-葡萄糖(TKDG；TDP-脱氧糖的中间体)生物利用度增加的基因突变，我们计划增加TKDG前体的积累。另外过量表达内源TKDG生物合成蛋白可能进一步优化TKDG的生产。最后，将外源TDP-脱氧糖生物合成基因导入菌株，将积累的TKDG池转化为特定的TDP-脱氧糖。具体地说，在第一阶段，我们将通过以下几个方面论证在大肠杆菌中生产TDP-脱氧糖的可行性：1)开发分离、纯化和鉴定大肠杆菌产生的TDP-脱氧糖的分析方法；2)通过代谢工程使大肠杆菌积累TKDG；以及3)通过生产各种TDP-脱氧糖来证明所产生的TKDG高产菌株的实用性。具体地说，负责单独生产TDP-D-岩藻糖、TDP-D-岩藻糖和TDP-D-橄榄糖的生物合成基因将被置于诱导表达载体中，并转化到积累TKDG的工程菌中。我们认为，在第一阶段的研究中，所产生的菌株将能够生产至少50 mg/L的TDP-D-岩藻糖、TDP-D-岩藻糖和/或TDP-D-橄榄糖。在第二阶段，我们将扩大TDP-脱氧糖的生产规模，并优化色谱技术以提高产量。此外，我们将继续利用各种TDP-脱氧糖生物合成基因来生产氨基糖、支链糖和额外的脱氧糖。在第三阶段，我们将把开发的技术商业化，提供多种TDP-糖出售，进行TDP-糖的定制合成，并实施定制的糖多样化项目。 公共卫生相关性：该项目的目标是开发大肠杆菌作为生产活性TDP-脱氧糖的全细胞生物催化剂。这些脱氧糖可以用来制造具有新治疗特性的天然产品的衍生物，例如，对抗生素耐药性细菌有效的抗生素。

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. We propose to investigate a novel approach to produce rare TDP-deoxysugars in Escherichia coli through metabolic engineering. By combining genetic mutations which separately lead to increased bioavailability of glucose-6-phosphate (G6P; an intermediate of TKDG) and TDP-4-keto-6-deoxy-D-glucose (TKDG; an intermediate of TDP-deoxysugars) we plan to increase the accumulation of the TKDG precursor beyond previous reports. Additional over-expression of endogenous TKDG biosynthetic proteins may further optimize TKDG production. Finally, exogenous TDP-deoxysugar biosynthetic genes will be introduced into the strain to convert the accumulated TKDG pool into specific TDP-deoxysugars. Specifically, in Phase I we will demonstrate the feasibility of TDP-deoxysugar production in E. coli through by 1) developing analytical methods for the isolation, purification and characterization of TDP-deoxysugars produced by E. coli, 2) metabolically engineering E. coli to accumulate the TKDG and, 3) demonstrating the utility of the resulting TKDG over-producing strain by producing various TDP-deoxysugars. Specifically, biosynthetic genes responsible for the individual production of TDP-D- fucose, TDP-D-fucofuranose and TDP-D-olivose will be placed into inducible E. coli expression vectors and transformed into the engineered strain accumulating TKDG. We propose the resulting strains will be capable of producing at least 50 mg/L of TDP-D-fucose, TDP-D-fucofuranose and/or TDP-D-olivose in the Phase I study. In Phase II, we will scale-up production of TDP-deoxysugars and optimize chromatographic techniques for increased throughput. Furthermore, we will continue to utilize various TDP-deoxysugar biosynthetic genes for the production of amino sugars, branched-chain sugars and additional deoxysugars. In Phase III we will commercialize the technology developed by offering a wide variety of TDP-sugars for sale, carrying out custom synthesis of TDP-sugars, and carrying out custom glycodiversification projects. PUBLIC HEALTH RELEVANCE: This project is aimed toward developing E. coli as a whole-cell biocatalyst for the production of activated TDP-deoxysugars. These deoxysugars can be used to make derivatives of natural products with new therapeutic properties, for example, antibiotics that are effective against antibiotic-resistant bacteria.