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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 wild-type UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l^-1 (1.6 times of wild-type UGTSL2) rebaudioside D from 20 g l^-1 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.

SUBMITTER: Chen L

PROVIDER: S-EPMC7264896 | biostudies-literature |

REPOSITORIES: biostudies-literature

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