Construction of immobilized enzyme cascades for the biosynthesis of nucleotide sugars UDP-N-acetylglucosamine and UDP-glucuronic acid

UDP-sugars, as active forms of monosaccharides, play integral roles in glycosylation and biosynthesis of polysaccharides. Although enzymatic catalysis has achieved great process, the comparatively low productivity and the time-consuming enzyme purification processes restricted its practical applications. Here, we developed two CipA-dependent enzyme immobilization systems for synthesis of UDP-GlcNAc and UDP-GlcA. Initially, we selected and identified the enzyme combinations of PpAmgK and SeGlmU for UDP-GlcNAc (3.19 mM) synthesis, and AtGlcAK and BlUSP for UDP-GlcA (1.83 mM) synthesis. After optimizing the molar ratios of substrates, the production of UDP-GlcNAc and UDP-GlcA increased to 17.33 mM and 9.03 mM when setting UTP:GlcNAc:ATP and UTP:GlcA:ATP as 1:1:1 and 1:2:1, respectively. Then, the polyphosphokinase SePPK for recycling ADP and PPi was introduced, resulting in a significant increase in UDP-GlcNAc (29.33 mM) and UDP-GlcA (20.87 mM). Eventually, the CipA-based immobilization systems were developed for repetitive catalysis. The combinations of PSK-(G₄S)₃-CipA and CipA-(G₄S)₃-ABK yielded the comparable productions of UDP-GlcNAc (28.66 mM, 17.40 g/L) and UDP-GlcA (20.34 mM, 11.80 g/L) within 75 min. This study presents a convenient and reusable CipA-based enzyme immobilization system for synthesis of UDP-sugars, showing great potential for enzymatic production of UDP-GlcNAc and UDP-GlcA.