Metabolic Engineering of a curdlan-producing Agrobacterium sp. for sugar nucleotide cofactor regeneration

农杆菌sp. ATCC 31749是一种工业菌株，用于生产凝乳素，一种同型葡萄糖聚合物。该菌株中葡萄糖聚合物的异常高产源于高效的udp -葡萄糖辅助因子再生机制。本项目提出利用这种自然进化的机制作为平台，开发高效的糖核苷酸辅助因子再生系统，用于酶促低聚糖合成。首先，聚合步骤可以通过引入凝乳酶合成酶基因的突变而中断。由此产生的突变体可以作为一个有效的UDP-葡萄糖再生系统。此外，利用udp -葡萄糖是其他三种糖核苷酸生物合成途径的切入点这一事实，研究计划提出了将转基因农杆菌菌株转化为udp -半乳糖、udp -葡萄糖醛酸和udp -半乳糖醛酸再生系统的策略。该项目还将通过新的有效的遗传和基于广泛宿主范围的异源蛋白表达方法，为代谢工程/生物催化研究做出贡献，适用于广泛的非e。杆菌的细菌。

Agrobacterium sp. ATCC 31749 is an industrial strain used to produce curdlan, a homo-glucose polymer. The unusually high productivity of the glucose polymer in this strain results from a highly efficient UDP-Glucose cofactor regeneration mechanism. This project proposes to use this naturally evolved mechanism as a platform to develop highly efficient sugar nucleotide cofactor regeneration systems useful for enzymatic oligosaccharide synthesis. First, the polymerization step can be disrupted by introducing mutations in the curdlan synthase gene. The resulting mutant can function as an efficient UDP- glucose regeneration system. Further, taking advantage of the fact that UDP-glucose is the entry point of the biosynthetic pathways of three other sugar nucleotides, the research plan proposes strategies to transform the genetically modified Agrobacterium strain into UDP-galactose, UDP-glucuronic acid and UDP-galacuronic acid regeneration systems. This project will also contribute to metabolic engineering/biocatalysis research through new and effective genetic and broad-host-range based heterologous protein expression methods applicable to a wide range of non-E.coli bacteria.