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A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50  = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations., (© 2024. The Author(s).)

In an attempt to reveal the relationships between alkaloid biosynthesis and phylogeny, we investigated by GC-MS the alkaloid patterns of 22 species and 3 hybrids (from 45 locations) from seven main sections of the genus Narcissus (Amaryllidaceae). The results indicate that the first alkaloids to evolve in the genus Narcissus were of the lycorine- and homolycorine-type. The alkaloid pattern of the Nevadensis section supports its recent separation from the Pseudonarcissus section. The plants of Narcissus pallidulus (Ganymedes section) show a predominance of Sceletium-type compounds, which are quite rare in the Amaryllidaceae family. Two successful evolutionary strategies involving alkaloid biosynthesis and leading to an expansion in taxa and occupied area were determined. Firstly, a diversification of alkaloid patterns and a high alkaloid concentration in the organs of the large Narcissus species (in the Pseudonarcissus section) resulted in an improved chemical defence in diverse habitats. Secondly, both plant size and alkaloid biosynthesis were reduced (in the Bulbocodium and Apodanthi sections) relegated to dry pastures and rocky places., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Narcissus pseudonarcissus (L.) cv. Carlton is being cultivated as a main source of galanthamine from the bulbs. After galanthamine, haemanthamine and narciclasine are the next most abundant alkaloids in this cultivar. Both these compounds are promising chemical scaffolds for potential anticancer drugs. For further research and drug development, a reliable supply of these compounds will be needed. In this study a field experiment was conducted to investigate the levels of galanthamine, haemanthamine and narciclasine in plants of N. pseudonarcissus cv. Carlton. In a field experiment alkaloids in the bulbs, leaves and roots were analyzed by quantitative (1)H NMR to monitor the variations during the growing season. Major primary and secondary metabolites were identified in the various plant parts. Multivariate data analysis was performed on the (1)H NMR spectra to investigate how metabolites changed in the plant organs over time. The results show that the leaves have relatively high concentrations of the alkaloids before flowering. The bulbs had lower concentrations of the compounds of interest but would have a higher total yield of alkaloids due to bigger biomass. Narcissus pseudonarcissus cv. Carlton represents a good source of galanthamine, and can potentially be a source of the other major alkaloids depending on choice of organ and harvest time., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Large-scale plant cultivation usually involves the use of pesticides. Apart from eliminating the target organism, the external chemicals may affect the metabolism of the crop plant. This may have implications for plants cultivated for specific medicinal compounds. In this study the effects of diverse fungicides on the metabolism of Narcissus pseudonarcissus cv. Carlton bulbs were investigated. N. pseudonarcissus cv. Carlton is being cultivated for the extraction of the alkaloid galanthamine. Fungicides typically used in Narcissus cultivation were applied in a field experiment. The aim was to determine whether fungicide applications changed the concentration of galanthamine in the bulbs. (1)H NMR spectroscopy allowed quantitative analysis of galanthamine and other metabolites in bulb extracts. Multivariate data analysis revealed changes in bulb metabolite patterns caused by fungicides. Bulbs treated before planting generally had higher levels of alkaloids, while foliar field applications caused lower alkaloid levels but altered carbohydrate metabolism. Within these groups, certain fungicide treatments caused changes in specific metabolites. This study shows that the fungicides used in Narcissus cultivation can cause a change in the metabolome still detectable in the bulbs after harvest. The standard cultivation practices in terms of fungicide treatment were found suitable for the production of N. pseudonarcissus cv. Carlton as raw material for galanthamine extraction. In the cultivation of medicinal plants for secondary metabolites the potential effect of pesticides and other agrochemicals should be taken into account., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Liquid-shake cultured shoot-clumps of Narcisus confussus were treated with the commonly used biotic elicitors methyl jasmonate, arachidonic acid, chitosan and salicylic acid. The effects of these compounds on the growth of the explants, as well as on the amount of the alkaloids released to the liquid culture medium and accumulated in the tissues at the end of the experiment were studied. The obtained results showed that, in general, high doses of these compounds had a negative effect on the growth of the explants, particularly the salicylic acid. On the contrary, the addition of methyl jasmonate, mainly at 25 microM, promoted the release of galanthamine and other related alkaloids to the liquid medium in proportions of up to 300% in relation to the control explants, and also their accumulation in tissues. The other elicitors studied did not have any interesting effects on the production of these Amaryllidaceae-type alkaloids.

Galanthamine is an Amaryllidaceae-type alkaloid with acetylcholinesterase inhibitory activity which is used in the treatment of Alzheimer's disease. The distribution of galanthamine and four other alkaloids in different organs of Narcissus confusus plants, as well as the variations occurring during the ontogenic cycle of this plant species, was studied. The five alkaloids were found to be present in all the organs at every stage, with the exception of haemanthamine in senescent flowers. The highest amount of alkaloids occurred in the bulb at the emerging stage, galanthamine being the most abundant, reaching a concentration of up to 2.5 % referred to dry weight.