Integration of a Nucleoside Triphosphate Regeneration System in the One-pot Synthesis of UDP-sugars and Hyaluronic Acid

Enzymatic synthesis is a promising approach to produce hyaluronic acid (HA) products with defined molecular weight (MW) and dispersity. We here report on the evaluation of nucleoside triphosphate (NTP) regeneration system for the synthesis of the precursors uridine-5'-diphosphate alpha-d-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphosphate alpha-d-glucuronic acid (UDP-GlcA) and integration in the one-pot synthesis of HA. By integration of polyphosphate kinase from Ruegeria pomeroyi (RpPPK2-3) into the enzyme cascade to synthesize UDP-GlcA we reached 89 % yield after 1 h with a regeneration cycle number (RCN) of 17. With the UDP-GlcNAc cascade, the ATP concentration was lowered 250-fold and reached 94 % yield after 1 h with an RCN of 234. Integration of the RpPPK2-3 regeneration system in one-pot HA synthesis afforded optimization of MgCl2 and ATP starting concentrations. With 25 mM MgCl2 and 0.1 mM ATP, a HA concentration of 0.81 g L-1 with an average MW of 1.17 MDa and dispersity of about 1.08 and an RCN of 75 was obtained. We conclude that integration of NTP regeneration with RpPPK2-3 expanded the enzymatic possibilities to produce HA with lower range MW.

Automated summary

The integration of RpPPK2-3 regeneration system expanded the enzymatic possibilities to synthesize HA with lower range molecular weight. The experiment results showed that high yields and optimized molecular weight and dispersity could be achieved under specific conditions.