This study describes the molecular characterization and heterologous expression of farnesyl pyrophosphate synthase (PsFPS) from Panax sokpayensis Shiva K. Sharma & Pandit. FPS is a key regulatory enzyme of ginsenoside biosynthetic pathway that directs the metabolic flux from mevalonate (MVA) and methylerythritol phosphate (MEP) pathway towards the synthesis of ginsenosides and phytosterols. Using Rapid Amplification of cDNA Ends (RACE), full-length PsFPS of 1437 bp with an open reading frame (ORF) of 1026 bp coding for 342 amino acids was cloned. Deduced amino acid sequence of PsFPS contained two highly conserved aspartate rich motifs with consensus DDXXD sequence that constitute the main catalytic site of FPS. Full length cDNA of PsFPS has 5′ untranslated region (UTR) with 142 bp and 3′ UTR with 234 bp followed by a poly (A) tail. In silico analysis detected three identical polyadenylation signals (PAS) with canonical sequence, AATAAA in the 3′ UTR. Alignment of 3′ UTR of PsFPS with the FPS from other Panax species indicated the presence of alternative PAS which could have possible role in the formation of different isoforms of FPS in Panax species. Open reading frame of PsFPS was expressed in Escherichia coli (E. coli) and the recombinant protein was produced in vitro. High expression of recombinant protein was observed after 1 h of induction with 1 mM IPTG. HPLC mediated enzyme assay confirmed the production of functional PsFPS in E. coli. This is the first report on molecular characterization, heterologous expression and functional validation of PsFPS from P. sokpayensis.

这项研究描述了法拉第焦磷酸合酶（PsFPS）的分子特征和异源表达的三七总生Shiva K.Sharma＆Pandit。FPS是人参皂苷生物合成途径的关键调节酶，可指导甲羟戊酸（MVA）和甲基赤藓糖醇磷酸酯（MEP）途径的代谢通量走向人参皂甙和植物甾醇的合成。使用cDNA末端快速扩增（RACE），克隆了1437 bp的全长PsFPS和1026 bp的开放阅读框（ORF），编码342个氨基酸。推导的PsFPS氨基酸序列包含两个高度保守的天冬氨酸丰富基序，具有共有的DDXXD序列，构成FPS的主要催化位点。的全长cDNAPsFPS具有142 bp的5'非翻译区（UTR）和234 bp的3'UTR，其后是poly（A）尾巴。在计算机分析中，在3'UTR中检测到三个具有规范序列AATAAA的相同的聚腺苷酸化信号（PAS）。将PsFPS的3'UTR与其他人参物种的FPS进行比对表明存在替代的PAS，这可能在人参物种中形成不同的FPS亚型中可能发挥作用。在大肠杆菌（E. coli）中表达PsFPS的开放阅读框，并在体外产生重组蛋白。。用1 mM IPTG诱导1小时后，观察到重组蛋白的高表达。HPLC介导的酶法测定证实在大肠杆菌中产生了功能性PsFPS 。这是在分子表征，异源表达和功能验证所述第一报告PsFPS从P. sokpayensis。