Abstract
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 β-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant β1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-β-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, β1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 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 β1,4 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 β1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides.
Key points
• A novel β-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9.
• EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis.
• Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.
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Acknowledgements
This work was supported in part by Projects of Science and Technology Department of Shandong Province (Project no. 2020CXGC010602), the National Key R&D Program of China (Project no. 2021YFC2103102), National Natural Science Foundation of China (Project no. 31770845), Open Projects Fund of NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, and Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology.
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JS designed and coordinated the work. Jiu-Ying Sun carried out the experiments. Jiu-Ying Sun and JD carried out the HPLC analysis. RD and ZL carried out the donor experiments. Jiu-Ying Sun, SX, and YC carried out the synthesis and purification of oligosaccharides. XG and FW conducted MS and NMR analyses. JS and Jiu-Ying Sun wrote the manuscript. All authors read and approved the final manuscript.
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Sun, JY., Deng, JQ., Du, RR. et al. Novel β1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides. Appl Microbiol Biotechnol 107, 5119–5129 (2023). https://doi.org/10.1007/s00253-023-12671-5
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DOI: https://doi.org/10.1007/s00253-023-12671-5