Development of In Vitro and In Planta Strategies for Efficient Production of High‐Value Steviol Glycosides

Steviol glycosides (SGs) are a group of sweet-tasting diterpene glycosides found in Stevia rebaudiana. Production of high-value SGs, Rebaudioside D (Reb D) and Reb M, is limited by inefficient conversion of Reb A to Reb D and stevioside to Reb E. To address this, we investigated the combined use of stevia UDP-glycosyltransferase SrUGT76G1 and a UDP-glycosyltransferase from Setaria italica (SiUGT) to enhance SG biosynthesis. SiUGT was previously shown to efficiently convert Reb A and stevioside to Reb D and Reb E, respectively, in yeast. Using an in vitro enzymatic assay, we demonstrated that combining SrUGT76G1 and SiUGT significantly improved conversion efficiency, with stevioside identified as a more favourable starting substrate due to reduced formation of unwanted byproducts. Subsequently, SrUGT76G1 and SiUGT were expressed individually and as a fusion protein in the heterologous host Nicotiana benthamiana via transient and stable transformation. While transient expression of either SrUGT76G1 or SiUGT in N. benthamiana resulted primarily in intermediate SGs being produced, transient expression of the SrUGT76G1-SiUGT fusion protein enabled production of both Reb D and Reb M upon substrate feeding. Likewise, stable transgenic N. benthamiana lines expressing the fusion construct SrUGT76G1-SiUGT accumulated Reb D and Reb M, with production levels correlating positively with the transgene expression. Overall, this study established an optimised in vitro enzymatic system for efficient Reb M production, as well as demonstrated the feasibility of N. benthamiana as an in planta platform for heterologous SGs biosynthesis, providing novel strategies with potential applicability for industrial production of high-value SGs.