莲花（Nelumbo nucifera）已被用作食物、药物和精神象征将近3000年。莲的药用特性主要归因于其苯基异喹啉生物碱（BIA）的独特配方，其中包括潜在的抗癌、抗疟疾和抗心律失常化合物。莲花的BIA生物合成与罂粟和其他毛茛目植物成员有着显著的区别，尤其是在拥有（R）-立体化学构型的BIA中具有丰富的含量和大多数BIA生产者的主要分支中间产物间丝距花碱的缺失。由于这些独特的代谢特征和药理学潜力的莲，我们开始阐明N. nucifera中的BIA生物合成网络。在此，我们展示了莲花CYP80G（NnCYP80G）和秘鲁肉豆蔻（Laurelia sempervirens）的优良同源物（LsCYP80G）立体选择性地将（R）-N-甲基可可啶转化为前芥茉碱生物碱格拉佐芬（glaziovine），随后甲基化为假阴离子素（pronuciferine），被认为是Nuciferine的前体。虽然神圣的莲采用一条专用的（R）-路径向有机化合物前芥茉碱生物碱的方向发展，但我们实施了人工立体化学反转方法，翻转了核心BIA途径的立体化学。利用罂粟的去氢间丝距花碱合成酶的独特底物特异性，将它与去氢间丝距花碱还原酶配对，从（S）-norcoclaurine开始，进行（R）-N-甲基可可啶的新生物合成，然后将其转化为假阴离子素。我们利用自己的立体化学反转方法， 阐明了莲花代谢中NnCYP80A的作用，表明其催化双BIA nelumboferine的立体选择性形成。我们筛选了66种植物O-甲基转移酶，并促进了从nelumboferine到潜在的抗癌bis-BIA liensinine的转化。我们的工作突显了N. nucifera独特的苯基异喹啉代谢，并利用经过改良的微生物系统来定向生产潜在的莲药物。Copyright © 2023. 版权所有Elsevier Inc.

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.Copyright © 2023. Published by Elsevier Inc.