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

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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.