TY - JOUR
T1 - Enhanced Production of Rebaudioside D and Rebaudioside M through V155T Substitution in the Glycosyltransferase UGT91D2 from Stevia rebaudiana
AU - Shoji, Tsubasa
AU - Tanaka, Yoshikazu
AU - Nakashima, Yu
AU - Mizohata, Eiichi
AU - Komaki, Maki
AU - Sugawara, Satoko
AU - Takaya, Junichiro
AU - Yonekura-Sakakibara, Keiko
AU - Morita, Hiroyuki
AU - Saito, Kazuki
AU - Hirai, Tadayoshi
N1 - Publisher Copyright:
© 2025 The Authors. Published by American Chemical Society.
PY - 2025/1/22
Y1 - 2025/1/22
N2 - Steviol glycosides (SGs) are noncaloric natural sweeteners found in the leaves of stevia (Stevia rebaudiana). These diterpene glycosides are biosynthesized by attaching varying numbers of monosaccharides, primarily glucose, to steviol aglycone. Rebaudioside (Reb) D and Reb M are highly glucosylated SGs that are valued for their superior sweetness and organoleptic properties, yet they are present in limited quantities in stevia leaves. This study aims to improve the substrate preference and catalytic efficiency of UDP-sugar-dependent glycosyltransferase UGT91D2 from stevia, which acts as a bottleneck in the biosynthesis of Reb D and Reb M. We modeled the structure of UGT91D2 and substituted two amino acid residues, Y134 and V155, which are located near the glycosyl acceptor and donor, respectively. Expression of the UGT91D2V155T in budding yeast significantly enhanced the production of Reb D and Reb M. Furthermore, transient expression in Nicotiana benthamiana revealed that the V155T substitution improved the glucosylation activity of UGT91D2, suggesting that this substitution enhances UDP-glucose binding and reduces side reactions involving nonglucose donors. By coexpressing multiple stevia UGT genes in N. benthamiana, we successfully produced highly glucosylated SGs from steviol. Our results provide insights into the substrate specificity of UGT91D2 and contribute to the engineering of SG biosynthesis.
AB - Steviol glycosides (SGs) are noncaloric natural sweeteners found in the leaves of stevia (Stevia rebaudiana). These diterpene glycosides are biosynthesized by attaching varying numbers of monosaccharides, primarily glucose, to steviol aglycone. Rebaudioside (Reb) D and Reb M are highly glucosylated SGs that are valued for their superior sweetness and organoleptic properties, yet they are present in limited quantities in stevia leaves. This study aims to improve the substrate preference and catalytic efficiency of UDP-sugar-dependent glycosyltransferase UGT91D2 from stevia, which acts as a bottleneck in the biosynthesis of Reb D and Reb M. We modeled the structure of UGT91D2 and substituted two amino acid residues, Y134 and V155, which are located near the glycosyl acceptor and donor, respectively. Expression of the UGT91D2V155T in budding yeast significantly enhanced the production of Reb D and Reb M. Furthermore, transient expression in Nicotiana benthamiana revealed that the V155T substitution improved the glucosylation activity of UGT91D2, suggesting that this substitution enhances UDP-glucose binding and reduces side reactions involving nonglucose donors. By coexpressing multiple stevia UGT genes in N. benthamiana, we successfully produced highly glucosylated SGs from steviol. Our results provide insights into the substrate specificity of UGT91D2 and contribute to the engineering of SG biosynthesis.
KW - UDP-glucose
KW - UDP-sugar-dependent glycosyltransferase
KW - stevia
KW - steviol glycoside
UR - http://www.scopus.com/inward/record.url?scp=85214335396&partnerID=8YFLogxK
U2 - 10.1021/acs.jafc.4c09392
DO - 10.1021/acs.jafc.4c09392
M3 - 学術論文
C2 - 39783863
AN - SCOPUS:85214335396
SN - 0021-8561
VL - 73
SP - 2019
EP - 2032
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
IS - 3
ER -