Abstract
BackgroundEnzymatic 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.ResultsTo 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%.ConclusionsThis 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.
Highlights
Glucuronic acid (GlcA) is one of the common building blocks of polysaccharides, proteoglycans and glycoglycerolipids [1]
uridine 5′-diphosphate (UDP)-glucuronic acid (GlcA) is one of nine nucleotide donor sugars used by glycosyltransferases in mammals
A number of glycosyltransferases using UDP-GlcA as sugar donor have been identified and prepared [11,12,13,14,15], which provides a guide to an efficient approach to prepare glycans containing GlcA residues [16,17,18,19]
Summary
Glucuronic acid (GlcA) is one of the common building blocks of polysaccharides, proteoglycans and glycoglycerolipids [1]. UDP-GlcA is one of nine nucleotide donor sugars used by glycosyltransferases in mammals It is biosynthesized via two Understanding and application of enzymatic glycan synthesis has leapt forward in recent years [5,6,7,8]. A number of glycosyltransferases using UDP-GlcA as sugar donor have been identified and prepared [11,12,13,14,15], which provides a guide to an efficient approach to prepare glycans containing GlcA residues [16,17,18,19]. 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 mono‐ saccharide donor for these glucuronidation reactions
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