Abstract：
Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose (G-G-R) sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton. Until recently, the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive. In this study, we identified two novel glycoside glycosyltransferases (GGTs) that can sequentially install the sugar chain\'s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside, two representative G-G-R sugar chain-containing triterpenoid saponins produced by Centella asiatica. Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase. We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families, suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order. Moreover, expression patterns of the two GGTs in pierced leaves of C. asiatica were found to be correlated with the production of asiaticoside and madecassoside, implying their involvement in host defense against herbivores and pathogens. Our work sheds light on the biosynthesis and evolution of complex saponin sugars, paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.
摘要：
Apiales顺序中的某些植物物种积累了三萜皂苷，其特征是独特的葡萄糖-葡萄糖-鼠李糖（G-G-R）糖链连接在五环三萜骨架的C-28位置。直到最近,糖链生物合成和进化的基因组基础仍然难以捉摸。在这项研究中,我们确定了两种新的糖苷糖基转移酶（GGT），它们可以在积雪草苷和羟基积雪草苷的生物合成过程中依次安装糖链的第二个D-葡萄糖和第三个L-鼠李糖，积雪草生产的两种代表性的含G-G-R糖链的三萜皂苷。酶测定揭示了两个GTT的显着底物混杂性以及对葡萄糖基转移酶和鼠李糖基转移酶的糖供体选择性至关重要的关键残基。我们进一步揭示了伞形科和Araliaceae家族中两个GGT的同质串联基因重复，这表明糖链组装具有保守的基因组基础，这可能是在阿皮亚莱斯家族的早期祖先中进化而来的。此外，发现两种GGT在积雪草的刺穿叶中的表达模式与积雪草苷和madecassososide的产生有关，暗示他们参与宿主对食草动物和病原体的防御。我们的工作揭示了复杂皂苷糖的生物合成和进化，为未来具有独特糖型的多种生物活性三萜类化合物的工程铺平了道路。
