Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera)

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3,000 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)-armepavine to pronuciferine, a proaporphine alkaloid and 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)-armepavine 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. SIGNIFICANCE We report the elucidation and microbial reconstitution of the aporphine (NnCYP80G) and bis-benzylisoquinoline (NnCYP80A) branches of alkaloid metabolism from sacred lotus (Nelumbo nucifera). Our findings demonstrate that both NnCYP80G and NnCYP80A are stereospecific for (R)-substrates. Linking an artificial stereochemical inversion pathway to a high-titer (S)-norcoclaurine production strain afforded a de novo route to the bis-benzylisoquinoline nelumboferine and the proaporphine pronuciferine via (R)-intermediates. We also devised a novel path to liensinine, a promising lotus chemotherapeutic, by screening 66 diverse methyltransferases for activity on nelumboferine. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.