Turning waste into treasure: Biosynthesis of value-added 2-O--glucosyl glycerol and D-allulose from waste cane molasses through an in vitro synthetic biology platform

The efficient and economical conversion of agricultural waste into glycosides and rare sugars is challenging. Herein, an in vitro synthetic bienzyme system consisting of sucrose phosphorylase and D-allulose 3-epimerase was constructed to produce 2-O-alpha-glucosyl glycerol and D-allulose from cane molasses. Lactic acid in the cane molasses significantly induced sucrose phosphorylase to hydrolyze sucrose instead of glycosylation. Notably, lactic acid significantly inhibited the catalytic performance of D-allulose 3-epimerase only in the presence of Na+ and K+, with an inhibition rate of 75%. After removing lactic acid and metal ions, 116 g/L 2-O-alpha-glucosyl glycerol and 51 g/L D-allulose were synthesized from 500 mM sucrose in the treated cane molasses with a sucrose consumption rate of 97%. Our findings offer an economically efficient and environmentally friendly pathway for the industrial production of glycosides and rare sugars from food industry waste.