Microbial Synthesis of Chondroitin Sulfates and N-Glycolyl Chondroitin Using Engineered Escherichia coli.

The glycosaminoglycan chondroitin and its derivatives, such as chondroitin sulfates, have important pharmaceutical applications. Escherichia coli K4 produces a chondroitin-like capsular polysaccharide. In this work, we demonstrate that this E. coli K4 can be engineered to synthesize three important derivatives of chondroitin. We demonstrate in vivo chondroitin-4-sulfate (CS-A) and chondroitin-4,6-sulfate (CS-E) production by the expression of the chondroitin-4-O-sulfotransferase enzyme and chondroitin 4-O-sulfo 6-O-sulfotransferase enzyme, respectively, and the accumulation of 3'-phosphoadenosyl-5’-phosphosulfate (PAPS). Using metabolic engineering and growth optimization, CS-A and CS-E with high sulfation levels were synthesized. Since this product is made entirely microbially in one-step, it is more sustainable and applicable in the pharmaceutical industry as well as cell-culture studies compared to current production methods that involve extraction from animal tissues.

Another chondroitin derivative synthesized in this study is N-glycolyl chondroitin (Gc-CN). N-glycolylneuraminic acid (Neu5Gc) is a sialic acid biosynthesized from the hydroxylation of N-acetylneuraminic acid (Neu5Ac). The enzyme responsible for this is CMP-Neu5Ac hydroxylase encoded by the CMAH gene. During evolution, humans have lost this gene and, therefore, cannot synthesize Neu5Gc. Although humans cannot synthesize Neu5Gc, this sialic acid can be incorporated through exogenous sources such as by the consumption red meat. This exogenous incorporation has been linked to many diseases such as cancer, atherosclerosis, and type-2 diabetes. A more stable metabolite of Neu5Gc called N-glycolyl chondroitin (Gc-CN) will be used to test this correlation. 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 carbon source supplemented with chemically synthesized N-glycolyl glucosamine allowing the incorporation of N-glycolyl into chondroitin.