Amaryllidaceae Alkaloids Biosynthesis Research Articles

Multiple NADPH-cytochrome P450 reductases from Lycoris radiata involved in Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs), such as galanthamine and lycorine, are natural products of Lycoris radiata possessing various pharmacological activities including anti-acetylcholinesterase, anti-inflammatory, and antitumour activities. Elucidating the biosynthesis of these special AAs is crucial for understanding their production and potential modification for improved clinical application, of which cytochrome P450 enzymes catalyse the formation of key alkaloid skeletons and subsequent modification processes, with the NAPDH cytochrome P450 reductases (CPRs) serving as essential redox partners. This study identified three CPRs, LrCPR1, LrCPR2, and LrCPR3, encoding 700, 697 and 695 amino acids, respectively, which belong to Class II CPRs. The LrCPRs reduced cytochrome c and ferricyanide in an NADPH-dependent manner, and their activities all followed the typical Michaelis-Menten curve. In yeast, the co-expression of LrCPRs and CYP96T6 produced the galantamine-like alkaloid namely N-demethylnarwedine, suggesting that they support the catalytic activity of CYP96T6. Quantitative analysis of the transcriptional expression profiles showed that LrCPRs were expressed in all the examined tissues of L. radiata, and their gene expression patterns are consistent with other genes that may be involved in the biosynthetic pathway of AAs, including cinnamate 4-hydroxylase and phenylalanine ammonia-lyase. Our study firstly provides the functional characterization of LrCPRs in L. radiata, which will contribute to the discovery of biosynthetic pathways and heterologous production of AAs.

Functional characterization of CYP96T1-like cytochrome P450 from Lycoris aurea catalyzing para-para' and para-ortho' oxidative coupling in Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs) are complex plant secondary metabolites possessing a wide range of biological activities. 4'-O-methylnorbelladine (4OMN) is the branchpoint intermediate for the entire AAs, and was the last common intermediate before AA pathway branches diverge. The cyclization of 4OMN by C-C oxidative coupling, which can afford para-para', ortho-para', and para-ortho' scaffold, was catalyzed by cytochrome P450 96T (CYP96T) family enzymes. To clarify the mechanisms involved in this controversial step, four CYP96T homologs (LauCYP96T1, LauCYP96T1-like-1, LauCYP96T1-like-2 and LauCYP96T1-like-3) were cloned from the full-length transcriptome of Lycoris aurea. All the four LauCYP96T are localized to endoplasmic reticulum. Functional analysis reveals that LauCYP96T1 and LauCYP96T1-like proteins display inverted regioselectivity for oxidative coupling of 4OMN, in which LauCYP96T1 and LauCYP96T1-like-2 dominantly afford para-para' scaffold, and LauCYP96T1-like-1 and LauCYP96T1-like-3 are responsible for para-ortho' scaffold formation. Using molecular homology modeling and docking studies, we predicted models for the binding of 4OMN to LauCYP96T, and identified two amino acid residues that might be responsible for the dominant changes in generated products of para-ortho' and para-para' oxidative coupling. Our results highlight the functional diversity and promiscuity of LauCYP96T enzymes and might provide valuable information for Amaryllidaceae alkaloid production.

Ascorbate peroxidase catalyses synthesis of protocatechualdehyde from p-hydroxybenzaldehyde in Lycoris aurea

Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is also a key intermediate in the synthesis of Amaryllidaceae alkaloids for supplying the C6-C1 skeleton. However, the biosynthesis of protocatechualdehyde in plants remains obscure. In this study, we measured the protocatechualdehyde contents in the root, bulb, scape and flower of the Amaryllidaceae plant Lycoris aurea (L’Hér.) Herb., and performed the correlation analysis between the protocatechualdehyde contents and the transcriptional levels of the phenolic oxidization candidate protein encoding genes. We found that a novel ascorbate peroxidase encoded by the contig_24999 in the L. aurea transcriptome database had potential role in the biosynthesis of protocatechualdehyde. The LauAPX_24999 gene was then cloned from the cDNA of the scape of L. aurea. The transient expression of LauAPX_24999 protein in Arabidopsis protoplasts demonstrated that LauAPX_24999 protein was localized in the cytoplasm, thus belonging to Class II L-ascorbate peroxidase. Subsequently, LauAPX_24999 protein was heterogenously expressed in Escherichia coli, and identified that LauAPX_24999 biosynthesized protocatechualdehyde from p-hydroxybenzaldehyde using L-ascorbic acid as the electron donor. The protein structure modelling and molecular docking indicated that p-hydroxybenzaldehyde could access to the active pocket of LauAPX_24999 protein, and reside at the δ-edge of the heme group while L-ascorbic acid binds at the γ-heme edge. To our knowledge, LauAPX_24999 is the first enzyme discovered in plants able to biosynthesize protocatechualdehyde from p-hydroxybenzaldehyde, and offers a competent enzyme resource for the biosynthesis of Amaryllidaceae alkaloids via synthetic biology.

Navigating Amaryllidaceae Alkaloids: Bridging Gaps and Charting Biosynthetic Territories - A Comprehensive Review.

Amaryllidaceae alkaloid (AAs) biosynthesis has garnered significant attention in recent years, particularly with the commercialisation of galanthamine as a treatment for the symptoms of Alzheimer's disease. A significant amount of research work over the last 8 decades has focused on the understanding of AA biosynthesis, starting from early radiolabelling studies to recent multi-omics analysis with modern biotechnological advancements. Those studies enabled the identification of hundreds of metabolites, the characterisation of biochemical pathway, an understanding of the environmental stimuli, and of the molecular regulation of these pharmaceutically and agriculturally important metabolites. Despite the numerous works there remain significant gaps in understanding their biosynthesis in Amaryllidaceae plants. As such, further research is needed to fully elucidate the metabolic pathway and facilitate their production. This review aims to provide a comprehensive overall summary of the current state of knowledge on AAs biosynthesis, from elicitation of transcription factors expression in the cell nucleus to alkaloid transport in the apoplast, and to highlight the challenges that need to be overcome for further advancement.

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3ʹ-hydroxylase (LaeC3′H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3′H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Unravelling the C-C and C-N coupling mechanism for the CYP96T1-catalyzed biosynthesis of Amaryllidaceae alkaloids

Plant cytochrome P450s (CYPs) comprise a vast family of enzymes intricately involved in the biosynthesis of natural products. However, the mechanistic understanding of the plant P450s’ catalysis is rather limited before. In this study, comprehensive simulations were performed to elucidate the C-C and C-N coupling mechanism involved in the biosynthesis of Amaryllidaceae alkaloids catalyzed by CYP96T1. Our simulations propose that the C-C coupling process follows a diradical mechanism. Especially, the proximal phenol OH group can be H-bonded to the active species of Cpd I, allowing the facile hydrogen atom transfer (HAT) from the phenol OH to Cpd I. In addition, the initial HAT disrupts the H-bond interaction between the proximal phenol group and the Cpd II species, enabling the approach of the distal phenol group for the second HAT, en route to the diradical species. The following para-para' C-C phenolic coupling reaction was observed to take place within the active site. In the context of the C-N coupling reaction, our calculations indicate that the reactions proceed via an Aza-Michael addition mechanism, facilitated by the acid-base catalysis provided by phosphate ions from the pH buffer. Notably, both the C1-N and C5-N coupling exhibit comparable energy barriers, resulting in the formation of the experimentally observed products 3 ((6R, 1S)-noroxomaritidine) and 4 ((6R, 5S)-noroxomaritidine). Our investigation has not only elucidated the mechanistic aspects of the C-C and C-N formation mechanism underlying the biosynthesis of Amaryllidaceae alkaloids, but also underscored the synergy between enzymatic transformations within P450 and non-enzymatic transformations within an aqueous solution during the biosynthesis of natural products.

Characterization of norbelladine synthase and noroxomaritidine/norcraugsodine reductase reveals a novel catalytic route for the biosynthesis of Amaryllidaceae alkaloids including the Alzheimer's drug galanthamine.

Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites with diverse pharmacological properties. Norbelladine is the entry compound in AAs biosynthesis and is produced from the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA). There are two reported enzymes capable of catalyzing this reaction in-vitro, both with low yield. The first one, norbelladine synthase (NBS), was shown to condense tyramine and 3,4-DHBA, while noroxomaritidine/norcraugsodine reductase (NR), catalyzes a reduction reaction to produce norbelladine. To clarify the mechanisms involved in this controversial step, both NBS and NR homologs were identified from the transcriptome of Narcissus papyraceus and Leucojum aestivum, cloned and expressed in Escherichia coli. Enzymatic assays performed with tyramine and 3,4-DHBA with each enzyme separately or combined, suggested that NBS and NR function together for the condensation of tyramine and 3,4-DHBA into norcraugsodine and further reduction into norbelladine. Using molecular homology modeling and docking studies, we predicted models for the binding of tyramine and 3,4-DHBA to NBS, and of the intermediate norcraugsodine to NR. Moreover, we show that NBS and NR physically interact in yeast and in-planta, that both localize to the cytoplasm and nucleus and are expressed at high levels in bulbs, confirming their colocalization and co-expression thus their ability to work together in the same catalytic route. Finally, their co-expression in yeast led to the production of norbelladine. In all, our study establishes that both NBS and NR participate in the biosynthesis of norbelladine by catalyzing the first key steps associated in the biosynthesis of the Alzheimer's drug galanthamine.

SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

Endophytic bacteria from in vitro culture of Leucojum aestivum L. a new source of galanthamine and elicitor of alkaloid biosynthesis

Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L. aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P. lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A1, abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P. lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis.

Transcriptome analysis of Leucojum aestivum and identification of genes involved in norbelladine biosynthesis.

Transcriptome analysis of Leucojum aestivum led to the identification of 50 key genes associated with Amaryllidaceae alkaloid biosynthesis including norbelladine synthase which localized in the cytosol and catalyzed norbelladine formation. The Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites, which are known for their biological activities. Although the general chemical reactions in the AA biosynthetic pathway have been proposed, the genes and enzymes of the pathway remain largely unstudied. All AAs are synthesized from a common precursor, norbelladine, by the condensation of tyramine and 3,4-dihydroxybenzaldehyde. The enzyme norbelladine synthase (NBS) which catalyzes the condensation reaction has only been characterized at a molecular level from one species, and the subcellular localizations have not been explored. Hence, the intracellular compartments wherein the AAs are biosynthesized remain unknown. In this study, a first comprehensive transcriptomic analysis of summer snowflake (Leucojum aestivum) was done to identify key genes associated with AA biosynthesis. Fifty orthologous genes were identified and deposited into GenBank. In addition, we identified and further characterized NBS from the transcriptome of L. aestivum and previously reported Narcissus papyraceus. Phylogenetic analysis showed that LaNBS, NpNBS1 and NpNBS2 shared high amino acid identity. The heterologous expression of LaNBS produced a recombinant protein with NBS activity. Bioinformatic prediction and C-terminal GFP tagging in transiently transformed Nicotiana benthamiana showed that LaNBS, NpNBS1 and NpNBS2 were likely localized to the cytosol which suggests that the AA biosynthesis starts in the cytosol. This study provides an Amaryllidaceae transcriptome that will be very helpful to identify genes for characterization studies in AA metabolism in planta or using heterologous systems. In addition, our study will facilitate the bioengineering of AA biosynthetic pathway in plants or in microorganisms.

Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production.

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L. aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

Relative expression of putative genes involved in galanthamine and other Amaryllidaceae alkaloids biosynthesis in Narcissus field and in vitro tissues.

The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310µg Gal/g FW) and bulb (980-1150µg Gal/g FW) than the culture derived samples; callus (1.0-7.0µg Gal/g FW) and bulblets (12-215µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpO4OMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.

Biosynthesis of alkaloids in Amaryllidaceae plants: a review

Amaryllidaceae alkaloids are a group of specialized metabolites found predominantly in the Amaryllidaceae plant family. Approximately 600 naturally occurring Amaryllidaceae alkaloids have been identified, many of which possess a variety of potent biological and pharmacological properties. However, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of neurodegenerative disorders such as Alzheimer’s disease, is currently on the market. Despite promising pharmacological potential, the clinical development of Amaryllidaceae alkaloids is hindered by limited commercial availability. In contrast to the large body of knowledge on the pharmacological and phytochemical aspects of Amaryllidaceae alkaloids, their molecular and physiological features are less explored. A better understanding of Amaryllidaceae alkaloid biosynthesis and metabolic regulation is crucial to take advantage of new metabolic engineering technologies for improving the efficiency and sustainability of plant or microbial Amaryllidaceae alkaloid production. Although there is still much to learn, over the past several years comparative transcriptomic and traditional biochemical approaches have led to significant advances in the identification of the molecular players involved in producing Amaryllidaceae alkaloids including enzymes from the shikimate and phenylpropanoid pathways. The identity of few Amaryllidaceae alkaloid biosynthetic enzymes and several genes encoding them has been reported. In this review, an overview of the current knowledge on the biosynthesis of Amaryllidaceae alkaloids is presented.

Quantitative Determination of Lycorine and Galanthamine in Different in Vitro Tissues of Narcissus tazetta by GC-MS

Narcissus spp. are of great importance to pharmaceutical industries because of biosynthesis of Amaryllidaceae alkaloids. N. tazetta with several varieties is a widespread species, native to the Mediterranean region with considerable distribution in Iran. In the present study, calli, roots and bulblets were regenerated from N. tazetta var. Meskin. In order to report alkaloid profile and quantify galanthamine and lycorine, methanolic extract of different in vitro tissues were subjected to alkaloid purification by SPE (solid phase extraction) method, followed by GC-MS analysis. In general, galanthamine and lycorine were detected in all in vitro tissues, while demethylmaritidine was only identified in bulblets. Bulblet was identified as the best source of main Amaryllidaceae alkaloids regarding the highest quantities of galanthamine and lycorine with 15 and 731 µg/g dry weight (DW) respectively.

The versatile O-methyltransferase LrOMT catalyzes multiple O-methylation reactions in amaryllidaceae alkaloids biosynthesis

Amaryllidaceae alkaloids are unique benzylphenethylamine derivatives that comprise of more than 600 members with a huge chemical diversity. Most of them showed interesting bioactivities, for instance, galanthamine (GAL) is clinically used for Alzheimer's disease treatment. All Amaryllidaceae alkaloids had been thought to be derived from 4′-O-methylnorbelladine originated from norbelladine catalyzed by norbelladine 4′-O-methyltransferase (N4OMT). Herein we mined the transcriptome datasets of Lycoris radiata, a GAL-producing plant. LrOMT was cloned, overexpressed in Escherichia coli, and purified to homogeneity. Bioinformatics analysis and enzymatic activity assays revealed that LrOMT is an S-adenosylmethionine-dependent Class I OMT. LrOMT exhibited both para- and meta-O-methylation activities toward norbelladine to give 4′- and 3′-O-methylnorbelladine. Twenty-four analogues, including the proposed biosynthetic intermediates, were introduced to investigate the substrate scope of LrOMT and it showed that the aromatic substrates should have two vicinal hydroxyl groups. The LrOMT-catalyzed O-methylation preference is dependent on the properties of the binding group of the substrates. The transcription levels of LrOMT were positively associated with the accumulation of the Amaryllidaceae alkaloids and the biosynthetic intermediates in L. radiata. The present work revealed that LrOMT catalyzes multiple O-methylation reactions and its characterization will be helpful to uncover novel biosynthetic genes for Amaryllidaceae alkaloids biosynthesis.

Developmental Regulation of the Expression of Amaryllidaceae Alkaloid Biosynthetic Genes in Narcissus papyraceus.

Amaryllidaceae alkaloids (AAs) have multiple biological effects, which are of interest to the pharmaceutical industry. To unleash the potential of Amaryllidaceae plants as pharmaceutical crops and as sources of AAs, a thorough understanding of the AA biosynthetic pathway is needed. However, only few enzymes in the pathway are known. Here, we report the transcriptome of AA-producing paperwhites (Narcissus papyraceus Ker Gawl). We present a list of 21 genes putatively encoding enzymes involved in AA biosynthesis. Next, a cDNA library was created from 24 different samples of different parts at various developmental stages of N. papyraceus. The expression of AA biosynthetic genes was analyzed in each sample using RT-qPCR. In addition, the alkaloid content of each sample was analyzed by HPLC. Leaves and flowers were found to have the highest abundance of heterocyclic compounds, whereas the bulb, the lowest. Lycorine was also the predominant AA. The gene expression results were compared with the heterocyclic compound profiles for each sample. In some samples, a positive correlation was observed between the gene expression levels and the amount of compounds accumulated. However, due to a probable transport of enzymes and alkaloids in the plant, a negative correlation was also observed, particularly at stage 2.

Exogenous melatonin stimulated Amaryllidaceae alkaloid biosynthesis in in vitro cultures of Leucojum aestivum L

Leucojum aestivum L. is a highly demanding pharmaceutical plant which is a natural source of pharmacologically active Amaryllidaceae alkaloids, especially galanthamine and lycorine. To establish an efficient method for the production of these alkaloids Leucojum aestivum plants derived from somatic embryos were subjected to various concentrations of melatonin (N-acetyl-5-methoxy tryptamine), NaCl and melatonin combined with NaCl. The maximum L. aestivum plant biomass was obtained when 10 μM of melatonin was applied to the medium. These plants were characterised by normal growth and did not reveal signs of browning. Enrichment of the medium with NaCl caused a reduction in plant biomass, plant pigment contents (chlorophyll a, chlorophyll b and carotenoids) and an increase in the activities of antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). The addition of melatonin to the medium containing NaCl neutralised the negative effect of salt stress. Moreover, the stimulating effect of melatonin on Amaryllidaceae alkaloids biosynthesis was observed. In plants grown in the medium enriched with melatonin the galanthamine content was, on average, 58.6 times higher, and the lycorine content was 1.5 times higher as compared with the control. In vitro plants obtained from the medium supplemented with melatonin also showed the highest distribution of alkaloids. It is noteworthy that only the plants grown in the medium with melatonin showed the presence of tazettine and chlidanthine, 1,2-dihydro-. This study presents a promising biotechnological approach for the commercial production of alkaloids, including galanthamine and lycorine.

Cloning and characterization of norbelladine synthase catalyzing the first committed reaction in Amaryllidaceae alkaloid biosynthesis

BackgroundAmaryllidaceae alkaloids (AAs) are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties. Previous investigations on AA biosynthesis have revealed that all AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. Specifically, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis.ResultsWith the availability of wild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes for a small protein of 19 kDa with an anticipated pI of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PR10/Bet v1 proteins and shared 41% amino acid identity to opium poppy NCS1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry.ConclusionsHere, we describe a novel enzyme catalyzing the first committed step of AA biosynthesis, which will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.

Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope.

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

Molecular Cloning and Characterization of a meta/para-O-Methyltransferase from Lycoris aurea.

O-methyltransferases (OMTs) have been demonstrated to play key roles in the biosynthesis of plant secondary metabolites, such as alkaloids, isoprenoids, and phenolic compounds. Here, we isolated and characterized an OMT gene from Lycoris aurea (namely LaOMT1), based on our previous transcriptome sequencing data. Sequence alignment and phylogenetic analysis showed that LaOMT1 belongs to the class I OMT, and shares high identity to other known plant OMTs. Also, LaOMT1 is highly identical in its amino acid sequence to NpN4OMT, a norbelladine 4′-OMT from Narcissus sp. aff. pseudonarcissus involved in the biosynthesis of Amaryllidaceae alkaloids. Biochemical analysis indicated that the recombinant LaOMT1 displayed both para and meta O-methylation activities with caffeic acid and 3,4-dihydroxybenzaldehyde, and showed a strong preference for the meta position. Besides, LaOMT1 also catalyzes the O-methylation of norbelladine to form 4′-O-methylnorbelladine, which has been demonstrated to be a universal precursor of all the primary Amaryllidaceae alkaloid skeletons. The results from quantitative real-time PCR assay indicated that LaOMT1 was ubiquitously expressed in different tissues of L. aurea, and its highest expression level was observed in the ovary. Meanwhile, the largest concentration of lycorine and galanthamine were found in the ovary, whereas the highest level of narciclasine was observed in the bulb. In addition, sodium chloride (NaCl), cold, polyethylene glycol (PEG), sodium nitroprusside (SNP), and methyl jasmonate (MeJA) treatments could significantly increase LaOMT1 transcripts, while abscisic acid (ABA) treatment dramatically decreased the expression level of LaOMT1. Subcellular localization showed that LaOMT1 is mainly localized in cytoplasm and endosome. Our results in this study indicate that LaOMT1 may play a multifunctional role, and lay the foundation for Amaryllidaceae alkaloid biosynthesis in L. aurea.