Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5′-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L−1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.

中文翻译：

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通过偶联表达超嗜热酶的多个全细胞的级联合成尿苷5'-二磷酸葡萄糖醛酸

近年来，酶促聚糖的合成已取得了飞跃发展，已鉴定并制备了多种葡萄糖醛酸转移酶（EC 2.4.1.17），这为制备包含葡萄糖醛酸（GlcA）残基的聚糖的有效方法提供了指导。尿苷5'-二磷酸（UDP）活化形式UDP-GlcA是这些葡萄糖醛酸化反应的单糖供体。为了以经济有效的方式生产UDP-GlcA，开发了一种有效的三步级联途径，使用表达超嗜热酶的全细胞从淀粉中制得UDP-GlcA。通过在单个菌株中将具有适当表达水平的辅酶再生系统与UDP-葡萄糖6-脱氢酶偶联，细胞能够满足NAD +的要求。在不添加外源NAD +的情况下，反应产生了1.3 g L-1 UDP-GlcA，分别代表UDP-Glc和UTP的100％和46％的转换。最后，开发了一种阴离子交换色谱纯化方法。已从级联系统成功获取UDP-GlcA。纯化期间UDP-GlcA的产率为约92.0％。这项工作在大肠杆菌宿主细胞中建立了从头开始的超高温生物合成级联反应，这些细胞能够满足NAD +辅因子的要求，并充当UDP-GlcA合成的微生物工厂，这为大规模生产更便宜的UDP-GlcA打开了一扇门。 。