Abstract
Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.
Highlights
Stevia rebaudiana (Bertoni) is a self-incompatible plant species, and one of a few species in the Stevia genus whose leaves produce and accumulate high quantities of sweet steviol glycoside (SG) compounds[1]
Since the original characterization of UDP-dependent glycosyltransferases (UGTs) of the steviol glycoside pathway[7], the number of steviol glycosides (SG) species known to exist within the Stevia plant has expanded greatly, as analytical technologies have enabled the detection of species found in relatively minor abundance
NTV1 displays a relatively balanced steviol glycoside profile (Supplementary Table 2), which is desirable for experiments designed to test the effects of SG pathway gene manipulation in influencing the flux of metabolites through the pathway
Summary
Stevia rebaudiana (Bertoni) is a self-incompatible plant species, and one of a few species in the Stevia genus whose leaves produce and accumulate high quantities of sweet steviol glycoside (SG) compounds[1]. More recent studies have yielded additional insights with respect to role of UGT76G1 in the production of SGs. Using in vitro assays of Saccharomyces cerevisiae-derived lysates, it was demonstrated that in addition to its well documented role in catalyzing the glucosylation of stevioside to produce Reb A, UGT76G1 was capable of transferring glucose groups to position 3′ of the C13 glucose of steviol-1,2-bioside, rubusoside and Reb E to produce Reb B, Reb G, and Reb D, respectively[8]. Knowledge of the structures of the minor SG species, combined with our current understanding of the substrate specificities of the existing characterized UGTs of the SG pathway enables one to predict the route of synthesis of these minor SGs. Steps of the SG pathway where the enzyme responsible has been predicted, but not experimentally validated, are shown in Fig. 1 as dotted lines. It is not entirely clear how many distinct isoforms of UGT76G1 exist
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