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.

中文翻译：

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新型 β1,4 N-乙酰氨基葡萄糖转移酶在透明质酸寡糖从头酶法合成中的应用

摘要

使用多杀巴斯德氏菌透明质酸合酶 (PmHAS ) 从头合成透明质酸 (HA) 的效率受到其在单糖作为受体底物的初始反应步骤中催化活性较低的限制。在这项研究中，我们鉴定并鉴定了源自大肠杆菌O8:K48:H9的 O 抗原基因合成簇的 β-1,4- N-乙酰氨基葡萄糖转移酶 (EcGnT)。当葡萄糖醛酸单糖衍生物4-硝基苯基-β-D-葡萄糖醛酸苷(GlcA- p NP)作为受体时，重组β1,4 EcGnT有效催化HA二糖的产生。与 PmHAS 相比，β1,4 EcGnT以 GlcA- p NP 作为受体表现出优异的N-乙酰氨基葡萄糖转移活性（约 12 倍），使其成为从头合成 HA 寡糖的第一步的更好选择。然后，我们开发了一种生物催化方法，使用 β1,4 EcGnT 产生的二糖作为起始材料合成尺寸控制的 HA 寡糖，然后逐步 PmHAS 催化合成较长的寡糖。使用这种方法，我们生产了一系列多达 10 个糖单体的 HA 链。总体而言，我们的研究鉴定了一种新型细菌 β1,4 N -乙酰氨基葡萄糖转移酶，并建立了一种更有效的 HA 寡糖合成工艺，可实现 HA 寡糖的尺寸控制生产。

关键点

• 来自大肠杆菌O8:K48:H9 的新型β-1,4-N-乙酰氨基葡萄糖转移酶(EcGnT)。

• EcGnT 在实现 HA 寡糖从头合成方面优于 PmHAS。

• 使用EcGnT 和PmHAS 进行尺寸控制的HA 寡糖合成中继。