Oriented efficient biosynthesis of rare ginsenoside Rh2 from PPD by compiling UGT-Yjic mutant with sucrose synthase

Ginsenoside Rh2 (3β-O-Glc-protopanaxadiol), a trace but an important pharmacological component of ginseng, has exhibited excellent medicinal potential. Many studies have found that the synthesis of Rh2 by UDP-glucosyltransferase (UGT) is an alternative production strategy. In this study, Yjic from B. subtilis 168 was found to synthesize ginsenoside F12 (3β,12β-Di-O-Glc-protopanaxadiol) and Rh2 at a ratio of 7:3. Yjic regioselectivity toward Rh2 synthesis was successfully improved using a semi-rational design including structure-guided alanine scanning and saturation mutations. As a result, mutant M315F was found to efficiently synthesize Rh2 (~99%) and block the further glycosylation of C12-OH. The circulation of UDPG was achieved by combining M315F with AtSuSy through a cascade reaction. Furthermore, an extraordinarily high yield of Rh2 (3.7 g/L) was attained in an aqueous solvent system with 17% DMSO (v/v) through the fed-batch feeding of PPD. This study presents the high potential for the oriented preparation of ginsenoside Rh2.