(594f) The Metabolic Engineering of E. coli for N-Glycolyl Chondroitin Production

In this study, we synthesized a derivative of chondroitin sulfate called N-glycolyl chondroitin sulfate (Gc-CS). This derivative is very valuable because it has medical and evolutionary applications. Gc-CS is hypothesized to be able to detect diseases like carcinomas, atherosclerosis, etc. This is because, according to recent studies, it was found to be a metabolite of one of the sialic acids that cause these diseases in humans. N-glycolylneuraminic acid (Neu5Gc) is a sialic acid biosynthesized from the hydroxylation of N-acetylneuraminic acid (Neu5Ac). The enzyme responsible for this hydroxylation is CMP-Neu5Ac hydroxylase encoded by the CMAH gene. During evolution, humans lost this gene and, therefore, cannot synthesize Neu5Gc. Although humans cannot synthesize Neu5Gc, it can be incorporated through exogenous sources such as eating red meat. Since humans don’t synthesize this sialic acid, the body recognized this as an antigen, and it recruits antibodies. This interaction triggers an inflammation that affects epithelial and endothelial tissues. To test this correlation between red meat consumption and the development of diseases in humans, a more stable metabolite of Neu5Gc called N-glycolyl chondroitin sulfate (Gc-CS) can be used. We have been able to synthesize chondroitin with this glycan modification using metabolically engineered E. coli K4 adapted for chondroitin production. The bacteria were fed with a glucose carbon source supplemented with chemically synthesized N- glycolyl glucosamine, and this allowed the incorporation of N- glycolyl into chondroitin. More recently we are focusing on the metabolic engineering of the E. coli strain to increase N-glycolyl incorporation into the cell. After exploring the pathway for chondroitin sulfate synthesis, we explored two avenues to shift the metabolic flux towards N-glycolyl incorporation. We explored the effect of downregulating the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase gene (GlmU) and the Glutamine-fructose-6-phosphate aminotransferase gene (GlmS).