CARB 92 |
| Research has shown the involvement of oligosaccharides in cancer, inflammation, cell-cell recognition, and other biological processes. This has led to the development of medical treatments involving specific oligosaccharide moieties. However, the production of these medical treatments is limited by oligosaccharide formation. Glycosyltransferase-catalyzed reactions have increased the selectivity and yield of the desired oligosaccharide, yet the high cost of the donor sugar nucleotides prohibits the large-scale production of oligosaccharides by this means. Through metabolic engineering of Agrobacterium sp., we have developed an effective UDP-galactose regeneration system. The naturally efficient UDP-glucose regeneration pathway of Agrobacterium sp. was extended by introducing a plasmid containing a UDP-galactose epimerase/galactosyltransferase fusion enzyme. This engineered strain synthesized approximately 18mM of UDP-galactose derived disaccharides. Unexpectedly, lactose was formed preferentially over n-acetyl-lactosamine (LacNAc), the desired galactose-containing product. Subsequently, various strategies were applied to enhance the specificity for LacNAc formation. The use of sucrose as a carbon source increased the ratio of LacNac to lactose from 0.053 to 0.77, yet surprisingly, the use of sucrose also led to the formation of a new galactose-containing sugar. Increasing the substrate concentration from 20mM to 100mM of n-acetyl-glucosamine further increased LacNAc:lactose to 3.96. A similar increase in LacNAc specificity resulted from using a fed-batch addition of glucose. These results demonstrate that metabolic engineering of the UDP-glucose pathway in Agrobacterium sp. is an effective means of producing UDP-galactose derived disaccharides. Based on this success, similar metabolic engineering strategies will be applied to generate other UDP-sugar regeneration systems. |
|
General Papers: Analytical Chemistry and Biochemistry of Carbohydrates and Derivatives
8:30 AM-12:30 PM, Thursday, 30 March 2006 Georgia World Congress Center -- B409, Oral
Division of Carbohydrate Chemistry |