Multienzyme Synthesis of Glycyrrhetic Acid 3-O-mono-β-d-glucuronide by Coupling UGT73F15 to UDP-Glucuronic Acid Regeneration Module

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG), a rare and innovative compound in licorice, exhibits high-potency sweetness and improved physiological activities. However, low amounts of GAMG from plants cannot meet the demands of growing markets. In this study, an efficient one-pot multienzyme cascade reaction for GAMG biosynthesis was constructed using a coupled catalysis of glycosyltransferase and uridine 5′-diphosphate (UDP) glucuronic acid (GlcA) regeneration system. The Glycyrrhiza uralensis glycosyltransferase UGT73F15 was expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of UGT73F15 were found to be pH 7.5 and 35 °C. The catalytic efficiency (kcat/Km) for glycyrrhetic acid (GA) was 2.14 min−1 mM−1 when using UDP-GlcA as sugar donor. To regenerate costly UDP-GlcA, the one-pot multienzyme cascade reaction including UGT73F15, sucrose synthase, UDP-glucose dehydrogenase, and lactate dehydrogenase was adopted to synthesize GAMG from GA on the basis of the UDP-GlcA regeneration system. By optimizing the cascade reaction conditions, the GAMG production successfully achieved 226.38 mg/L. Our study developed an economical and efficient one-pot multienzyme cascade method for facile synthesis of GAMG and other bioactive glucuronosides.