Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni

Stevia rebaudiana (stevia) contains commercially important steviol glycosides, stevioside and rebaudioside A, these compounds have insulinotropic and anti-hyperglycemic effect. Steviol, stevioside and rebaudioside-A have taste modulation and insulin potentiation activity. Stevia leaves are composed of steviol (2-5%), stevioside (4-13%) and rebaudioside-A (1-6%). Stevioside has after-taste bitterness, rebaudioside-A is sweetest in taste among all the glycosides present. Therefore, lower ratio of rebaudioside-A to stevioside has bitter after-taste, which makes stevia plants unpalatable. By over-expressing the genes, SrUGT76G1 and SrKO, we propose to increase the ratio of RebA to stevioside in stevia. Various lines were generated and amongst them, seven lines had both the transgenes present. Co-overxpresion of SrUGT76G1 and SrKO led to the increased concentration of RebA in all the seven transgenic lines (KU1-KU7) than control plant and RebA to stevioside ratio also increased significantly. Steviol, stevioside and RebA showed a differential concentration in all the seven lines, but the pattern was the same in all of them and the ratio of RebA to stevioside increased dramatically. In transgenic line 2 (KU2), RebA showed a steep increase in concentration 52% the rebaudioside-A to stevioside ratio increased from 0.74 (control) to 2.83. In overall all the lines, RebA showed a positive correlation with steviol and stevioside. Overexpression of SrKO led to an increase in steviol which increased the stevioside, overexpression of SrUGT76G1 ultimately increased RebA concentration. In conclusion, concentration of RebA increased significantly with co- overexpression of SrUGT6G1 and SrKO genes. Lines with increased RebA are more palatable and commercially viable.