Fusion glycosyltransferase design for enhanced conversion of rebaudioside A into rebaudioside M in cascade

Rebaudioside M (RM) is an attractive target for developing next-generation sweeteners due to its exceptional sweetness, up to 350 times sweeter than sucrose, and its non-caloric nature. However, its low natural abundance makes the traditional plant extraction method neither cost-effective nor environmentally sustainable. This study presents a fusion enzyme design of UGT76G1 and UGT91C1, both UDP-dependent glycosyltransferases (UTGs), to produce RM from naturally abundant rebaudioside A in a cascade reaction. The fusion enzyme was produced by Pichia pastoris with a yield of ∼25 mg purified protein per liter of culture, displaying optimal activity at ∼42°C and pH 7.0. It was found that a peptide linker consisting of three repeats of GGGGS enhanced substrate channeling and the synthesis rate of RM by 1.8-fold. Intracellular overexpression of the fusion enzyme in P. pastoris cells demonstrated higher catalytic efficiency, thermostability, and recyclability than mixed cells expressing UGT76G1 or UGT91C1 separately, or mixed purified enzymes. This study provides a promising approach for the industrial preparation of RM.