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

PLOS ONE RESEARCH ARTICLE Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni Nazima Nasrullah ID1*, Javed Ahmad1, Monica Saifi1, Irum Gul Shah2, Umara Nissar3, Syed Naved Quadri1, Kudsiya Ashrafi1, Malik Zainul Abdin1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Nasrullah N, Ahmad J, Saifi M, Shah IG, Nissar U, Quadri SN, et al. (2023) Enhancement of diterpenoid steviol glycosides by cooverexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni. PLoS ONE 18(2): e0260085. https://doi.org/10.1371/journal. pone.0260085 Editor: Allah Bakhsh, Nigde Omer Halisdemir University, TURKEY Received: April 8, 2021 Accepted: November 2, 2021 Published: February 6, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0260085 1 Department of Biotechnology, CTPD, School of Chemical and Lifesciences, Jamia Hamdard, New Delhi, India, 2 Division of Genetics, IARI- Indian Agricultural Research Institute, New Delhi, India, 3 Department of Biomedical Research, University of Bern, Bern, Switzerland * (MZA); (NN) Abstract 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. Copyright: © 2023 Nasrullah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1. Introduction Data Availability Statement: The GC-MS file is available through github (https://github.com/ naizma/GC-MS-STEVIA-REBAUDIANA). This is the Among the natural sweeteners reported so far, steviol glycosides (SGs) are 200–300 times sweeter than sucrose [1]. They are present in Stevia rebaudiana Bertoni (Stevia), a perennial herb from the family Asteraceae native to eastern Paraguay, Barazil. Typically, these compounds include PLOS ONE | https://doi.org/10.1371/journal.pone.0260085 February 6, 2023 1 / 24 PLOS ONE Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO/SrUGT76G1 genes in Stevia rebaudiana raw GC-MS data from which we generated Heatmap, correlation table and other pics in the online web based interface MetaboAnalyst. Original gel images are uploaded as S1 Raw Images. Funding: This study was funded by DSR(SAP)University grants commission New Delhi-India to Professor M.Z.Adin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. stevioside (4–13% dry weight), steviolbioside (trace), the rebaudiosides, including rebaudioside A (RebA) (1–6%), rebaudioside B (trace), rebaudioside C (1–2%), rebaudioside D (trace), rebaudioside E (trace), rebaudioside F (trace) and dulcoside A (0.4–0.7%) [2–4]. After final nod from the food safety authorities around the world, steviol glycosides are being used in commercial beverages, confectionaries and foods worldwide. Demand of stevia is hence increasing day by day. SGs modulate taste responses and insulin release by activating TRPM5 (Transient receptor potential cation channel subfamily M member 5) ion channel, and the potentiation of TRPM5 activity protects mice against the development of high-fat diet induced hyperglycaemia. Thus stevioside, RebA and their aglycon steviol are novel contenders for the development of antidiabetic drugs [5]. Methylerythritol Phosphate Pathway (MEP) and the Mevalonate Pathway (MVA) pathways are the principal contributors of sesquiterpenes, whereas mono- and diterpenes, in particular SGs, are exclusively formed by the MEP pathway [6]. Like all diterpenes, steviol is synthesized from geranylgeranyl diphosphate (GGDP). The step in gibberellic acid (GA) biosynthesis is catalyzed by ent-kaurene oxidase (KO) leading to the synthesis of ent-kaurenoic acid from entkaurene before the branch point to steviol production [7]. Ent-kaurene oxidase in Arabidopsis, Cucurbita maxima and pea are expressed in developing seeds and young, rapidly growing tissues with low expression levels in mature leaves. In stevia, however, Ent-kaurene oxidase (SrKO) expresses in both young and mature leaf tissues [8]. Stevia SrKO is highly expressed in leaves, succulent stems, flowers and seedling shoots. Gene expression of SrKO has a positive correlation with the concentration of stevioside in stevia leaves [9]. UDP-glucosyltransferase 76G1 (SrUGT76G1) catalyses the conversion of stevioside to RebA via one-step glycosylation reaction, which increases the amount of sweet-tasting RebA and decreases the amount of stevioside that has a bitter aftertaste [10]. SrUGT76G1 has glycosylating activity, not only towards aliphatic and branched alcohols but also towards (substituted) phenols, flavonoids, gallates and glycosides. SrUGT76G1 have remarkably broad specificity, which can be exploited for the glucosylation of a wide range of acceptor molecules [1 (...truncated)