Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Abstract

Summary Steviol glycosides ( SG s) are extracted from Stevia leaves for use as a natural sweetener. Among SG s, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP ‐glycosyltransferase 76G1 ( Sr UGT 76G1 ) that is known to convert stevioside to Reb A through 1,3‐β‐ d ‐glucosylation in vitro . Interestingly, by overexpressing Sr UGT 76G1 , the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type ( WT ) plants up to 1.55 in transgenic lines without any significant changes in total SG s content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged Sr UGT 76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by Sr UGT 76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of Sr UGT 76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.

An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function

“…A skewing of the dulcoside A:Reb C ratio is also evident, though not as dramatic given that Reb C content is normally much greater than dulcoside A in control plants. The effects of 76G1-AY overexpression on the stevioside:Reb A, and dulcoside A:Reb C ratios in this study are very consistent with similar transgenic plant results recently reported 9 . These four SG compounds were the only ones analyzed in that study, however, due to limitations in their detection of the less abundant SG species.…”

“…Subsequent induction of callus and the maintenance of callus under Hyg selection was also conducted in the dark until regeneration. Very recently, researchers reported using tissue cultured leaves as the explant and successfully obtained stable transgenic Stevia plants 9,26 ; this suggests that both types of originating explant are suitable for efficient transformation. Similar to our results, this same research group also concluded that a prolonged period of dark incubation was critical for obtaining high efficiency Stevia transformation.…”

“…This suggests that the amino acid polymorphisms that exist between these two closely related isoforms alter the breadth of the enzyme's substrate specificity. Previous studies involving UGT76G1 have largely focused on the 76G1-AY isoform [7][8][9] . In the one study that investigated the 76G1-GQ variant (referred to therein as UGTSr), the authors were unable to recover activity from E. coli extracts, and instead relied on in vitro assays using extracts from tobacco suspension cultures expressing 76G1-GQ 28 .…”

“…Furthermore, successful recovery of transgenic Stevia callus, but not whole plants, was documented using a GUS-bar construct delivered via particle bombardment 25 . Very recently, a research group from the University of Singapore described the stable transformation of Stevia using kanamycin as the selectable marker and GFP as a reporter gene 9,26 .…”

Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations

“…The hydroxyl groups of steviol are glycosylated by the enzymes uridine diphosphate (UDP)-glycosyltransferases (UGTs), and the number of sugars attached by UGTs generates the various SGs ( Brandle & Telmer, 2007 ). The UGT74G1 enzyme is involved in the conversion of steviolbioside to stevioside, while the UGT76G1 enzyme converts stevioside into rebaudioside A ( Kim et al, 2019 ).…”