Dai Longhai, Liu Can, Zhu Yueming, Zhang Jiangsheng, Men Yan, Zeng Yan, Sun Yuanxia
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
Plant Cell Physiol. 2015 Jun;56(6):1172-82. doi: 10.1093/pcp/pcv043. Epub 2015 Mar 9.
Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.
罗汉果甜苷是从罗汉果果实中分离出的主要生物活性成分，属于葫芦烷型四环三萜皂苷家族，在全球范围内用作高效甜味剂，并具有多种显著的药理活性。罗汉果甜苷在体内由2,3-氧化角鲨烯通过一系列由葫芦二烯醇合酶（CbQ）、细胞色素P450（P450s）和尿苷二磷酸糖基转移酶（UGTs）催化的反应合成。然而，相关基因迄今尚未得到表征。在本研究中，我们报告成功鉴定了SgCbQ和UGT74AC1，它们先前是通过对罗汉果果实的RNA测序（RNA-seq）和数字基因表达（DGE）谱分析预测的。通过在缺乏羊毛甾醇合酶的酵母菌株GIL77中表达对SgCbQ进行功能表征，发现其仅积累葫芦二烯醇作为唯一产物。UGT74AC1作为His标签蛋白在大肠杆菌中异源表达，通过体外酶活性测定，它通过将葡萄糖部分转移到C-3羟基上形成罗汉果苷IE，显示出对罗汉果醇的特异性。本研究首次报道了从罗汉果中鉴定出CbQ和糖基转移酶。结果还表明，RNA-seq与DGE谱分析相结合，是发现参与三萜皂苷生物合成的候选基因的一种有前途的方法。
