Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi‐enzyme system

Abstract

Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi-enzyme reaction system with S. rebaudiana UDP-glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two-step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side-product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wildtype UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l À1 (1.6 times of wild-type UGTSL2) rebaudioside D from 20 g l À1 stevioside after reaction for 24 h. This system is useful for large-scale rebaudioside D production and expands our understanding of the pathways involved in its synthesis.

β-Glucosidase and β-Galactosidase-Mediated Transglycosylation of Steviol Glycosides Utilizing Industrial Byproducts

“…Moreover, they can use low-cost donors, such as sucrose, reaching product yields over 90% (Ko et al, 2016;Devlamynck et al, 2019;Son et al, 2020). Regarding plantderived UDP-glucosyltransferase-mediated bioconversion, the regeneration of UDP sugar donors with sucrose synthase is necessary, but yields over 70% can be obtained (Wang et al, 2016;Chen et al, 2021Chen et al, , 2020Chen et al, , 2018. Although all the above reported yields are undoubtedly higher than those reported in the present work, the glycosyl hydrolases used offer the advantage of forming β-1,4 glycosidic bonds, which cannot be broken by human digestive enzymes.…”

The Potential of Cold Plasma and Electromagnetic Field as Stimulators of Natural Sweeteners Biosynthesis in Stevia rebaudiana Bertoni

“…Increased demand for stevia products forces the search of new methods for enhancement of economical and ecological production. Different methods and their combinations to increase SG yield are used: selection and breeding of cultivars with high concentrations of SGs, micropropagation [ 6 ], optimization of cultivation conditions (photoperiod length, moisture, temperature) [ 8 ], fertilization including biofertilizers [ 9 ], treatment with nanoparticles [ 10 , 11 ], and modifying after-harvest procedures such as drying, extraction conditions, and enzymatic conversion of Stev to RebA [ 12 , 13 , 14 ]. The main disadvantages of these methods often are time-consuming, expensive, or polluting, growing conditions that are climate-dependent and therefore, are not easily modified.…”

Co-immobilized recombinant glycosyltransferases efficiently convert rebaudioside A to M in cascade

“…The optimal pH for the free and immobilized enzymes was 7, which was similar to other glycosyltransferases. 14,44 The immobilized UGT2 showed 53-73% of relative activity at pH 6.5, 7.5 and 8.5, while the immobilized UGT1 retained 80% activity in the range of 6.5-8.5, suggesting that the immobilized UGT1 displayed a higher catalytic activity over a wide pH range of 6.5-8.5. Therefore, the temperature of 37 C and pH 7.0 were chosen for the following studies.…”

“…1,4 Recently, the biosynthesis of Reb D and Reb M in vivo or in vitro has attracted widespread interest. 5,6 The biotransformation reactions of SGs in S. rebaudiana are catalyzed by a series of uridine diphosphate glucose (UDPG)dependent glycosyltransferase (UGTs), [6][7][8] e.g., UGT91D2 for biotransformation of Reb A to Reb D, and UGT76G1 for Reb D to Reb M. 9,10 Similarly, the engineered enzymes EUGT11 from Oryza sativa L. and UGTSL2 from Solanum lycopersicum can also catalyze the biotransformation of Reb A to Reb D. [9][10][11][12][13][14] Since Reb M is of much higher commercial value than that of Reb D, and its industrial production mainly comes from Reb D, 1 which has a relatively high cost and low solubility. It is of great promise to develop a one-pot cascade reaction to produce Reb M directly from a cheap substrate such as Reb A to reduce the cost.…”