Stevia rebaudiana is a plant under the Asteraceae family and has been reported as a healthier alternative to sugar. Steviol glycosides (SGs) is the group of secondary metabolites responsible for the sweet taste. Among nine SGs synthesised by S. rebaudiana, stevioside and rebaudioside A are the sweetest. The biosynthetic pathway of SGs partly involves conversion of geranylgeranyl diphosphate (GGDP) into steviol, catalysed by ent- kaurene synthase (KS), ent-copalyl diphosphate synthase (CPPS), and kaurene oxidase (KO). This study focuses on in silico molecular characterization and phylogenetic analysis of KS from Malaysia’s S. rebaudiana MS007 variety (Stevia MS007). The transcriptomic dataset of S. rebaudiana accession MS007 was used in initial experiment toward analysing the KS. Through the blastx homology search using transcriptomic dataset query Cluster-31069.42907, the Stevia rebaudiana kaurene synthase (SrKS) sequence was identified with the highest similarity percentage identity (99.62%). The protein domain prediction using InterPro yields IPR005630 (terpene synthase metal-binding domain) at positions 490 to 755 and IPR001906 (terpene synthase-N-terminal-domain) at positions 258 to 477. Multiple sequence alignment was conducted using MUSCLE and MEGA-X as phylogenetic tree analysis tool for constructing the phylogenetic analysis tree. Based on the bootstrap value from the phylogenetic analysis, Cluster-31069.42907 represents relationships between the ancestors. Since both Helianthus annuus and S. rebaudiana are Asteraceae species, the bootstrap value for both species was 100%. In conclusion, this research contributes to a better understanding of Stevia MS007 KS via in silico analysis.

Stevia rebaudiana is a plant of the Asteraceae family that is used as a natural sweetener. Stevia has been shown to be safe for human consumption and has been utilised as a sweetener substitute for diabetic and obese people. In this study, the structure and gene content involved in the synthesis of putative UDP-glycosyltransferase 76G1 (UGT76G1) protein in S. rebaudiana MS007 were analysed using an in-silico method. Homologous search using BlastP revealed the highest percentage of identity, score, and E-value for UDP-glycosyltransferase 76G1-like of Helianthus annuus (ID: XP_021973845.1). The presence of IPR002213 UDP-glucuronosyl/UDP-glucosyltransferase entry, which is available at locations 89bp to 246 bp, was also verified by the protein family search using InterPro. MEGA-X software was used to construct a molecular phylogeny study, revealing that this protein belongs to the Asteraceae family. To predict the primary, secondary, and tertiary protein structures of the putative UGT76G1 protein, the ProtParam, ExPasy, PSIPRED, and Phyre2 programmes were implemented. The putative UGT76G1 protein’s tertiary structure prediction was given a score of 100.0% confidence by the single highest scoring template and a coverage of 98%, with the dimension of the model being (Å) of X: 52.453, Y: 61.270, and Z: 48.102. The UGT76G1 model fulfilled the quality standards and was approved for further analysis after validation performed by PROCHECK, VERIFY3D, and ERRAT. Thus, the findings of this work have contributed to a better knowledge of putative UDP-glycosyltransferase 76G1 features and target recognition processes, which will lead to better information on protein-protein interaction in S. rebaudiana MS007. Keywords: Phylogenetic, Stevia rebaudiana, UGT76G1, 3D Structure Prediction