Novel β1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a beta-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant beta 1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-beta-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, beta 1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (similar to 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by beta 1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial beta 1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides.