Citrate Stimulates Oligosaccharide Synthesis in Metabolically Engineered Agrobacterium sp.

Agrobacterium sp. ATCC 31749 was previously shown to be an advantageous host for oligosaccharide production. Unexpectedly, the addition of citrate to the oligosaccharide synthesis reaction resulted in up to a sixfold improvement in the production N-aceytl-lactosamine, a disaccharide. The possible mechanisms for this citrate-induced stimulation of oligosaccharide production were investigated, including the consumption of citrate as a carbon and energy source, enhanced metal ion solubility from citrate chelation, and the ability of citrate to act as a buffer. The main mechanisms for the effect of citrate on oligosaccharide production were determined to be carbon and energy provision from citrate consumption and pH maintenance. ATCC 31749 was shown to co-metabolize citrate along with sucrose, a preferred carbon source, indicating the lack of a catabolite repression system in this Agrobacterium. Metabolic flux analysis suggested an increase in flux through TCA cycle for the citrate-containing reaction, which may provide additional energy supply to support enhanced oligosaccharide production. The citrate stimulation of oligosaccharide synthesis was shown to be unique to the Agrobacterium strain, as a similarly engineered Escherichia coli strain did not show significant improvement in oligosaccharide production with citrate addition. This work provides insight into the metabolism of Agrobacterium sp. ATCC 31749 and highlights important factors in whole-cell oligosaccharide synthesis.