Metabolic engineering of Agrobacterium sp strain ATCC 31749 for production of an α-Gal epitope

Background: Oligosaccharides containing a terminal Gal-alpha 1,3-Gal moiety are collectively known as alpha-Gal epitopes. alpha-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the alpha-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the alpha-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the alpha-Gal epitope, Gal-alpha 1,3-Lac. Results: Agrobacterium sp. ATCC 31749 was engineered to produce Gal-alpha 1,3-Lac by the introduction of a UDPgalactose 4'-epimerase:alpha 1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the alpha-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting alpha-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-alpha 1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-alpha 1,3-Lac. Conclusions: The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of alpha-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the alpha-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.