Biological production of rebaudioside M with improved solubility, taste, and stability

Overview

This research proposes a biological production route for Rebaudioside M (RM), a steviol glycoside sweetener with enhanced solubility, pH stability, and taste quality compared to Rebaudioside A (RA). RM contains additional glucose units connected by β-glycosidic bonds, which improve hydrophilicity, inhibit crystallization, and prevent stimulation of bitter taste receptors due to its larger molecular size. Because RM occurs naturally in stevia leaves at very low concentrations, enzymatic conversion from RA offers a commercially viable production pathway. The approach addresses limitations of existing steviol glycoside sweeteners, including poor aqueous solubility (around 1% for RA), bitter aftertaste, and limited acidic stability seen in α-linked alternatives.

Technical specifications

Production system:

• Two bacterial expression platforms have been constructed: E. coli Top10 with pUC-28 plasmid and Bacillus subtilis WB600 with pHT-43 plasmid
• Three key enzymes are co-expressed: UDP76G1 (β-1,3 transglycosidase), UDP91D2 (β-1,2 transglycosidase), and sucrose synthase from potato for UDP-glucose regeneration
• IPTG induction triggers enzyme expression at optimal growth stage
• Reaction converts RA substrate into RM through sequential glucosylation

Key performance findings:

• UDP76G1 demonstrated activity converting 60% of stevioside to RA within 10 hours
• β-glycosidic linkages provide superior acidic resistance compared to α-linked GSG produced by starch-based enzymes
• Optimal induction temperature identified at 10°C to support correct protein folding
• Sorbitol supplementation under evaluation to enhance protein folding

Current challenges being addressed:

• Inclusion body formation in E. coli reducing enzyme productivity
• IPTG concentration insufficient for B. subtilis due to thicker cell wall

Technology readiness level

The technology is currently at laboratory-scale validation (TRL 3-4). Two expression systems have been successfully constructed and genes introduced, with initial enzyme activity confirmed. Optimization of expression conditions, including culture composition, medium feeding, inducing reagent concentration, and induction timing, is underway. Planned next steps include scale-up fermentation, enzyme purification (simplified in the B. subtilis system), RM synthesis testing, HPLC purity characterization, and stability and solubility validation. The project is estimated to take one year to complete. The research team has established partnerships within the stevia industry through membership in the China Stevia Organization and access to complete equipment at Jiangnan University plus a pilot line at Jiangsu Svetia Biotechnology Co. Ltd.