Dual-enzyme coupling system containing glycosyltransferase and sucrose synthase for efficient biotransformation of Rebaudioside A to Rebaudioside D in deep eutectic solvent system

E. coli P8 expressing a glycosyltransferase from Panax ginseng was constructed. • Coupling glycosyltransferase and sucrose synthase (SS) was adopted to yield Reb D. • Regeneration of UDPG in situ from UDP and sucrose was used to convert Reb A to Reb D. • DES (ChCl:glycerol=1:2) –H 2 O (4:6, v/v) enhances the conversion of Reb A to Reb D. • P8 cell and SS could convert 19.3 g/L Reb A to Reb D (21.0 g/L) in 8 h. Rebaudioside A (Reb A) can be converted into Rebaudioside D (Reb D) through a glycosylation reaction. Reb D is a higher-value sweetener with a very low caloric value, a glycemic index of zero, and greater sweetness than Reb A. However, its naturally low abundance limits large-scale production. Accordingly, this study developed an efficient bioconversion system for Reb D production. Two recombinant Escherichia coli strains were constructed and designated E. coli P8 and E. coli SS. E. coli P8 expressed a glycosyltransferase derived from Panax ginseng , whereas E. coli SS expressed a sucrose synthase derived from Arabidopsis thaliana . In the coupled system, sucrose synthase used sucrose and UDP to regenerate UDP-glucose (UDPG) in situ, while the glycosyltransferase catalyzed the conversion of Reb A into Reb D. A deep eutectic solvent (DES) composed of choline chloride and glycerol at a ChCl:Gly molar ratio of 1:2 was mixed with water to form a DES–H 2 O reaction medium. Under the selected conditions of pH 7.5, 45 °C, and a DES-to-water volume ratio of 40:60, P8 whole cells and an SS crude lysate converted 19.3 g/L Reb A into 21.0 g/L Reb D within 8 h using 500 mmol/L sucrose and 10 mmol/L UDP as co-substrates, corresponding to a molar yield of 93%. This study demonstrates an efficient and potentially environmentally friendly strategy for producing Reb D and other steviol glycosides. Download: Download high-res image (402KB) Download: Download full-size image