Construction of a Functional CMP-Sialic Acid Biosynthesis Pathway in Arabidopsis

Previous studies have reported that plants contain negligible amounts of free or protein-bound N -acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N -glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N -glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho- N -acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis ( Arabidopsis thaliana ) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase, N -acetylneuraminic acid phosphate synthase, and CMP- N -acetylneuraminic acid synthetase. Simultaneous expression of UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase and N -acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g −1 fresh weight in leaves) in planta, which could be further converted to cytidine monophospho- N -acetylneuraminic acid (2.4 nmol g −1 fresh weight in leaves) by coexpression of CMP- N -acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.