Efficient Production of Neohesperidin Enabled by Protein Engineering of Rhamnosyltransferase Cm1,2RhaT

Neohesperidin dihydrochalcone, a natural sweetener used extensively in food processing, is predominantly derived from neohesperidin hydrogenation. However, its production is challenged by low neohesperidin content in plants and limited enzyme activity of rhamnosyltransferase Cm1,2RhaT, vital for converting hesperetin 7-O-glucoside to neohesperidin. To augment Cm1,2RhaT's catalytic activity, we used a hybrid of structure-based semirational design and error-prone PCR to engineer a mutant, 49A/50ACm1,2RhaT, a 136-fold increase in catalytic efficiency toward hesperetin 7-O-glucoside. MD simulations and QM/MM computations suggest the enhanced enzymatic activity likely stems from the formation of an optimal substrate pocket that promotes substrate–ligand interaction and decreases reaction energy requirements. Using 49A/50ACm1,2RhaT, neohesperidin production peaked at 7.63 g/L. Furthermore, we established Cm1,2RhaT could expedite rhamnose transfer from UDP-rhamnose to the C-2 hydroxy in not just flavanones and flavones' 7-O-glucose, but chalcones and isoflavones' 7-O-glucose. This pioneering study provides new prospects for scalable neohesperidin production and effective biocatalysts for synthesizing valuable flavonoids.