Surface display of glycosyltransferase PgM8 and whole-cell catalysis for efficient Rebaudioside D biosynthesis in Pichia pastoris

Rebaudioside D (Reb D) is a zero-calorie, high-intensity sweetener favored for its superior taste profile compared to other steviol glycosides such as Stevioside (ST) and Rebaudioside A (Reb A). However, Reb D naturally accounts for only about 0.5% of the dry leaf mass of stevia, creating a production challenge. To address this, a mutated glycosyltransferase PgUGT (M8) (named PgM8) from Panax ginseng and sucrose synthase mbSUS from Vigna radiata were co-expressed in Pichia pastoris. We enhanced the system by fusing PgM8 with the GPI- anchored protein GCW61 for cell surface display, achieving enzyme immobilization. Optimizing the PgM8 copy number increased catalytic activity by 82.56%. This innovation enabled continuous whole-cell catalysis for Reb D synthesis, eliminating the need for cell disruption and purification while improving strain reusability. The yield of Reb D reached 48.2 g/L (42.7 mM) in a 50 mL batch within 33 hours, suggesting that this whole-cell catalyst has great potential for large-scale industrial production.