Engineering E. Coli Whole-Cell Catalyst for Efficient Synthesis of Lactosyl Oligosaccharide
Zichao Mao1, Anne Ruffing2, Hyun-Dong Shin2, Jeong S. Oh1 and Rachel Ruizhen Chen3, (1)School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Dr. NW, Atlanta, GA 30332-0100, (2)Chemical and Biomolecular Engineering, Georgia Institute of Technology, 778 Atlantic Dr., Atlanta, GA 30332, (3)Chemical&Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, NW, Atlanta, GA 30332-0100
Many valuable oligosaccharides contain lactose in its base structure. Examples include xenoplantation antigen alpha-Gal, and a cancer antigen Globo H. Developing carbohydrate based therapies such as anticancer vaccines are severely hampered by the difficulties in synthesizing these oligosaccharide structures. Previously we have shown that engineered E. coli whole-cells expressing requisite glycosyltransferase with cofactor (sugar nucleotide) regenerations are effective catalysts for complex carbohydrates. However, significant challenges still remain and metabolic engineering strategies will have to be devised to generate more efficient method. Using alpha-Gal trisaccharide (Gal-alpha1,3-Gal-beta1,4-Glc) as model compound, we investigated several engineering strategies to derive more efficient whole-cell catalysts.
Glycosyltransferase enzymes are group transfer enzymes, catalyzing the transfer of a monosaccharide from its activated form (sugar nucleotide or donor;) to an acceptor sugar. Since the donor sugars are high-energy compounds, their formation requires considerable metabolic energy. A whole-cell catalyzed reaction requires a sugar as an energy/carbon source while another acceptor sugar (in this case, lactose) needs to be taken up by the cells simultaneous with the uptake of the glucose. This requirement necessitates deregulation of catabolite repression, which prevents the uptake of lactose in the presence of glucose. Two strategies were investigated to overcome the problem. One is to use cells expressing a mutated form of cAMP receptor protein (CRP), another is to co-express lactose permease with other required enzymes.
Additional metabolic engineering strategies were also introduced including overexpression of uridine diphosphate kinase for enhanced flux in the regeneration of UDP-galactose, and melA gene ( encoding alpha-galatosidase) knockout to eliminate product degradation.
Collectively, these strategies led to a significantly improved synthesis. This presentation will highlight the new engineering strategies and compare various constructs in the synthesis. Overall, this study shows the effectiveness of metabolic engineering strategy in oligosaccharide synthesis.