Sustainable Production of Rebaudioside D via a Dual-Enzyme Cascade Utilizing Sucrose Synthase and Immobilized Glycosyltransferase

Rebaudioside D (Reb D) is a natural, zero-calorie, high-intensity sweetener. However, its low natural content in stevia leaves limits large-scale production. Conventional biotechnological methods often use expensive commercial sugars as carbon and energy sources, raising production costs and limiting scalability. To address these issues, this work developed a sustainable and efficient bioprocess for Reb D bioproduction. First, the biomass-hydrolysates acquired from low-cost and abundant agricultural waste are rich in glucose, serving as an efficient carbon source in microbial fermentation. The biomass-hydrolysates system constructed based on this can reduce costs, shorten reaction duration, and enhance enzymatic activity. In response to the current problems of the Reb D glycosylation enzyme system, such as low efficacy, poor stability, and reliance on expensive sugar donors like UDP-glucose, this work innovatively employed the biomass-hydrolysates fermentation strain GSS (sucrose synthase from Arabidopsis thaliana) for the first time to synthesize UDPGs from UDP, and fusing the Panax ginseng glycosyltransferase M8 with Saccharomyces cerevisiae Sed1p homologue (SED1) enabled its immobilization on the cell wall of Pichia pastoris via SED1. Afterward, a dual-enzyme system composed of GSS and M8 (activity ratio of 2:1) was constructed, utilizing the inexpensive sucrose (100 mM) to facilitate UDP recycling, enhancing the supply of UDP-glucose at 40 °C, pH 7.5, ultimately achieving a 96% Reb D yield. Through whole-cell catalysis, 1.08 × 103 mg/L Reb D was produced, demonstrating significant industrial application potential. In summary, this study introduced a promising dual-enzyme cooperative strategy for efficient Reb D synthesis. The use of a glycolysis medium highlights the potential to convert agricultural waste into high-value products.