Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.
Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
Plant Biotechnol J. 2019 Jun;17(6):1037-1047. doi: 10.1111/pbi.13035. Epub 2018 Dec 19.
Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, 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 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3-β-d-glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, 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 SGs 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 SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. 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 SrUGT76G1 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.
甜菊糖苷（SGs）是从甜菊叶中提取出来的，用作天然甜味剂。在 SGs 中，甜菊苷在叶提取物中最为丰富，其次是瑞鲍迪苷 A（Reb A）。然而，Reb A 因其与甜菊苷相比具有更甜、更可口的味道而备受关注。因此，开发具有更高 Reb A 与甜菊苷比例的新型甜菊品种将有利于生产更高质量的天然甜味剂。在这里，我们通过过表达已知在体外通过 1,3-β-d-葡萄糖基化将甜菊苷转化为 Reb A 的甜菊 UDP-糖基转移酶 76G1（SrUGT76G1），生成了转基因甜菊植物。有趣的是，通过过表达 SrUGT76G1，Reb A 与甜菊苷的比例从野生型（WT）植物中的 0.30 急剧增加到转基因系中的 1.55，而总 SGs 含量没有任何显著变化。这是由于 Reb A 含量的增加和甜菊苷含量的减少共同作用的结果。此外，我们还发现转基因系中 Reb C 与杜尔可苷 A 的比例增加。使用谷胱甘肽 S-转移酶标记的 SrUGT76G1 重组蛋白进行体外葡糖基转移酶测定，我们进一步证明 Reb C 可以通过 SrUGT76G1 从杜尔可苷 A 的葡萄糖基化产生。具有更高 Reb A 与甜菊苷比例的转基因甜菊植物在外观上与 WT 植物无法区分。总之，我们的结果表明，在甜菊中过表达 SrUGT76G1 是一种有效的方法，可以在不影响植物发育的情况下，生成具有更高比例更受欢迎的 Reb A 的新型甜菊品种。
