(213d) Metabolic Engineering of Agrobacterium Sp. for Synthesis of Complex Carbohydrate Epitopes

Carbohydrates play important roles in numerous biological processes, including many disease-causing events. They are often found on cell surfaces covalently linked to lipids or proteins. Many are capable of eliciting an immune response and are therefore known as epitopes. Two examples are N-acetyllactosamine (LacNAc) and α-gal epitopes containing a terminal Galα1,3Galβ. LacNAc is a disaccharide basal structure found in many antigens overexpressed on the surface of cancerous cells, while α-gal epitopes are responsible for hyperacute rejection of xenotransplantations. Cancer therapy and prevention of xenotransplantation rejection demand a high-purity and large-quantity supply of these complex carbohydrate epitopes.

Metabolic engineering is a promising approach for addressing the challenges in synthesizing complex carbohydrates. Previously, we successfully engineered a UDP-galactose regeneration system from the highly efficient UDP-glucose synthesis pathway in Agrobacterium sp., providing the precursor (UDP-galactose) for the formation of LacNAc. The engineered strain produced over 20 mM of the UDP-galactose derived disaccharides, LacNAc and lactose.

Additional metabolic engineering efforts were made to improve the synthesis. The Agrobacterium host strain was a natural producer of curdlan, a glucose polymer synthesized using UDP-glucose as precursor. Thus, curdlan production directly competes with LacNAc synthesis for UDP-glucose. Deleting the curdlan synthase gene, crdS, should allow the natural supply of UDP-glucose to be devoted solely for synthesis of the target oligosaccharide, yielding increased LacNAc synthesis in the engineered strain. The curdlan synthase gene deletion was successfully carried out by homologous recombination and confirmed through PCR and phenotypic studies. Significant improvement in LacNAc synthesis has been observed in preliminary investigation of the knockout strain.

For synthesis of the α-gal epitope Galα1,3Galβ1,4Glc, a gene fusion strategy was adopted to enhance product synthesis. A UDP-galactose 4'-epimerase was linked to a truncated, bovine α1,3-galactosyltransferase. This fusion enzyme consumes the endogenous UDP-glucose of Agrobacterium sp., converts it to UDP-galactose, and forms an α1,3-linkage between galactose and the acceptor, lactose. Synthesis of this α-gal trisaccharide presents an additional challenge as it requires simultaneous uptake of both sucrose (the preferred sugar and the carbon/energy source in the synthesis) and lactose (the disaccharide acceptor). To overcome catabolite repression that prevents lactose uptake, a lactose permease (lacY) from E. coli was co-expressed with the fusion enzyme, and the resulting strain was used for synthesis of the α-gal trisaccharide.

In this presentation, we detail the metabolic engineering strategies employed in these systems and highlight the results from various constructs. With these engineered strains, LacNAc and α-gal epitope synthesis may soon be possible at large scales. Furthermore, similar application of the strategies outlined in this work may enable production of other medically-relevant carbohydrates.