Reaction to Achieve A High Yield Stevia Rebaudioside D (Reb D) from Reb A

The Optimization of Reaction Conditions to Achieve A High Yield of Stevia Rebaudioside D

High-sugar diets have led to a rising prevalence of various chronic illnesses, including obesity, diabetes, cardiovascular diseases, and hypertension, posing a significant global public health concern. Stevia Rebaudioside D (Reb D), a natural sweetener derived from plants, has gained significant attention as a sugar substitute due to its zero-calorie content, intense sweetness, and pleasing taste profile.
However, its limited presence in stevia rebaudiana Bertoni and challenges related to low solubility and enzymatic activity of plant-derived glycosyltransferase have hindered its widespread commercial application. In response to these issues, a novel glycosyltransferase called YojK, originating from Bacillus subtilis 168, was discovered. This study introduces a genetically modified bacterial glycosyltransferase, YojK-I241T/G327N, characterized by high solubility and catalytic efficiency, offering great potential for industrial-scale production of Reb D.

The development of a highly efficient method for the production of rebaudioside D using a cascade reaction of YojK-I241T/G327N and AtSuSy enzymes.

To address the issue of low solubility in plant-derived glycosyltransferases, researchers investigated the potential of using YojK, an O-glycosyltransferase from Bacillus subtilis 168. YojK was chosen because of its demonstrated glycosylation activity with various substrates, including large molecules. After successful recombinant expression and purification, YojK was employed to glycosylate Reb A, resulting in the production of Stevia Reb D. Enzyme kinetic analysis revealed that YojK has lower catalytic activity toward Reb D compared to other glycosyltransferases. Consequently, the study suggests the necessity for structure-guided engineering to enhance YojK's catalytic activity for potential practical applications. (as shown in Figure 1)

stevia ra to stevia rd mechanism
Figure 1

The optimization of reaction conditions to achieve a high yield of rebaudioside D.

The reaction conditions, including pH, temperature, and substrate concentration, can be optimized to achieve a high yield of Srevia Reb D. The study used molecular dynamics simulations to investigate how the YojK-I241T/G327N variant enhances Reb D glycosylation. The variant maintained a stable hydrogen bond between Reb A and H14 during simulations, unlike the wild-type(as shown in Figure 2). This stability also extended to the distance between Reb A's O2 atom and UDPG's C1P. The YojK-I241T/G327N variant's improved catalytic efficiency was attributed to these stable interactions, suggesting its practical potential.

large yield production of stevia rd
Figure 2 

Researchers utilized a cascade reaction involving YojK-I241T/G327N and AtSuSy enzymes. Key optimization steps included maintaining pH at 8.0 in a potassium phosphate buffer, a reaction temperature of 35°C, 10% co-solvent DMSO, and a sucrose concentration of 400 mM.(as shown in Figure 3)

different conditions likely ph transformation
Figure 3


This approach yielded 83.47% Reb D. Notably, adding 1 mM UDPG allowed the synthesis of 20.59 g/L Reb D with a remarkable 91.29% yield, surpassing previous studies. This process demonstrates the potential for industrial-scale Reb D production, especially after structural engineering of YojK.
YojK can be efficiently expressed in E. coli BL21 (DE3) with high solubility. Through structure-guided engineering, a double mutant, YojK-I241T/G327N, was created, enabling the large-scale production of Reb D with an exceptional yield of 91.29% by recycling UDPG catalyzed by sucrose synthase AtSuSy. This study presents YojK-I241T/G327N as a promising tool for economically effective industrial-scale Reb D production. 


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