- 10:20 AM

Metabolic Engineering of E. Coli for Alpha-Gal Epitope Synthesis

Zichao Mao1, Anne Ruffing2, and Rachel Ruizhen Chen1. (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

Oligosaccharides containing alpha-gal epitope could be used in conjunction with organ transplantation to avoid or alleviate immune rejection. However, this requires an efficient synthesis method for the complex carbohydrate molecule, which has been a long-standing problem. Due to catabolite repression, the uptake of LacNAc or lactose (disaccharide acceptor sugars to which alpha-gal is to be attached) by cells in the presence of glucose is a daunting and unique challenge in the synthesis of alpha-gal-containing oligosaccharides. In this study, a metabolic engineering strategy was successfully applied to overcome the difficulty. An E. coli strain was first engineered to overexpress the two key enzymes (Pgm and GalU) involved in the UDP-glucose synthesis, which led to an enhanced carbon flux (approximately 8 fold more) to the UDP-glucose synthesis. By further metabolic engineering through introducing UDP-galactose 4' epimerase and a beta-1,4 galactosyltransferase, either LacNAc or lactose or both could be synthesized, with the ratio being dependent on the carbon source used and other conditions. This provides a mechanism to produce these two acceptor sugars in-situ so that the uptake difficulties could be circumvented. This will be demonstrated by introducing to the engineered strain a second galactosyltransferase enzyme, alpha 1,3-galactosyltransferase from a mammalian source, thus conferring the strain the ability to synthesize alpha-Gal containing trisaccharides. The presentation will detail the metabolic engineering strategies and a systematic search for the optimal flux to the desired products. As LacNAc and lactose are two disaccharides common to many other valuable oligosaccharides, the strategy of making both in situ is highly useful in complex carbohydrate synthesis.