Biology subjects

O'Connor, S. E.

Publications and source records attributed to O'Connor, S. E..

At least 19 recordsLinked to original sources

bioRxiv2026

An auxiliary protein tunes reductase activity in alkaloid biosynthesis

Monoterpene indole alkaloids encompass a diverse class of plant natural products. Many of these valuable compounds, including the anticancer medicines vinblastine and vincristine (Catharanthus roseus) and the antiaddiction treatment ibogaine (Tabernanthe iboga) utilize a common biosynthetic intermediate, 19E-geissoschizine, which is produced by the medium-chain dehydrogenase/reductase geissoschizine synthase (GS). Herein we report the discovery of a non-catalytic auxiliary protein (facilitator of geissoschizine synthase, FoGS) that improves formation of 19E-geissoschizine by acting in combination with GS. The discovery of a FoGS orthologue that works with GS to produce 19Z-geissoschizine shows how this protein also controls the stereochemistry of the product. We demonstrate that FoGS and GS interact to form a stable heterodimer, and show through mutagenesis that FoGS likely modulates the structure of the GS active site. Microscopy studies suggest that FoGS also changes the subcellular localization of GS. The discovery of FoGS will enable efforts to heterologously reconstitute commercially important geissoschizine-derived monoterpene indole alkaloids, and moreover, highlights how metabolic pathways utilize proteins that are not essential for production of the products, but nevertheless enhance the efficiency and specificity of the catalytic biosynthetic pathway components.

biochemistry↗

bioRxiv2026

Discovery of a Plant Pictet-Spenglerase with R-Stereoselectivity

The class-defining monoterpenoid indole alkaloid (MIA) scaffold strictosidine is generated by condensation of tryptamine and secologanin by the Pictet-Spenglerase strictosidine synthase (STR). All previously characterized STR orthologs are strictly 3S-stereoselective. Here, we report that the medicinal plant species Pogonopus speciosus (Rubiaceae) accumulates the 3R epimer vincosidic acid produced by a an ortholog of STR. This ortholog, named here Epi-STR, exclusively produces the 3R configuration and is capable of accepting both secologanic acid and the methylester secologanin as aldehyde substrates. Using comparative phylogenetic and structural analyses, we determine the amino acid residues that confer stereoselectivity. Through rational design of reciprocal amino acid substitutions, we achieved switches in stereoselectivity in a canonical STR and in Epi-STR. The stereoselectivity of the engineered mutants is also dependent on the identity of the aldehyde substrate. Notably, previous and extensive engineering efforts have failed to switch the stereo-selectivity of STR. Therefore, this discovery now allows cost-effective epimer-pure access to the R-epimer, and offers mechanistic insights into the enzyme stereoselectivity of this important reaction. This work also highlights the importance of phytochemical analyses of poorly described plant species.

biochemistry↗

bioRxiv2026

The floral illusion: A parasitic beetle mimics the scent of flowers to attract bees

Animals are not known to biosynthesize floral signals to manipulate pollinators, although such mimicry could profoundly shape plant-pollinator interactions. Larvae of the poisonous European blister beetle Meloe proscarabaeus parasitize multiple solitary bee species, yet the mechanism enabling host attraction has remained unresolved. Here we show that these larvae lure bees by emitting a bouquet of volatile compounds that closely resembles floral scent. Chemical analyses reveal a complex blend of monoterpenoids derived from (S)-linalool, a ubiquitous floral volatile. Behavioral assays demonstrate that these compounds function as floral cues, eliciting attraction in bees. Transcriptomic and functional analyses identify cytochrome P450 enzymes that oxidize (S)-linalool, demonstrating that larvae biosynthesize these plant-like volatiles de novo. Together, these findings broaden the scope of interkingdom chemical mimicry and uncover a striking form of sensory deception in which an insect chemically assumes the signal identity of a flower, revealing that animals can evolve biosynthetic pathways to exploit plant-pollinator communication.

ecology↗

bioRxiv2025

Conserved early steps of stemmadenine biosynthesis

Stemmadenine acetate is a pivotal intermediate in the production of pharmacologically active monoterpene indole alkaloids. Here, we identify orthologs of stemmadenine acetate pathway genes (SGD, GS, GO, Redox1, Redox2, SAT) from Tabernanthe iboga. We characterize these enzymes in vitro, and additionally, we reconstitute stemmadenine acetate biosynthesis in Nicotiana benthamiana, comparing the formation of intermediates and shunt products that are produced when previously characterized orthologs from the related plant Catharanthus roseus are used. Ortholog pairs are catalytically indistinguishable, except in the case of GS. Surprisingly, the T. iboga ortholog catalyzes formation of an alternate stereoisomer 19Z-geissoschizine, seeding a low-flux Z-series in heterologous reconstitution systems in vitro and in planta. We additionally characterize the major shunt products that arose during reconstitution of stemmadenine acetate biosynthesis. We show that the substrate promiscuity of Redox2 results in formation of the shunt products 16(R/S)-isositsirikines, hampering pathway flux and yields. Additionally, we show that stemmadenine can be oxidized by endogenous N. benthamiana enzymes, leading to the shunt product condylocarpine. Nevertheless, we could produce stemmadenine at a 6 mg yield from 19E-geissoschizine by heterologous expression in N. benthamiana. Overall, we highlight the prospects for milligram production of important biosynthetic intermediates in N. benthamiana.

biochemistry↗

bioRxiv2025

Discovery of iridoid cyclase completes the iridoid pathway in asterids

Iridoids are specialized monoterpenes ancestral to asterid flowering plants (Albach et al, 2001; Stull et al, 2018). Iridoids play key roles in plant defense and are also essential precursors for pharmacologically important alkaloids (Dobler et al, 2011; Eisner, 1964). The biosynthesis of all iridoids involves the cyclization of a reactive enol intermediate. While this cyclization occurs spontaneously at low yields, it has long been hypothesized that a dedicated enzyme is involved in this process (Geu-Flores et al, 2012; Lichman et al, 2019b). Here, we report the discovery of asterid iridoid cyclases (ICYC). We show that these enzymes catalyze cyclization of the reactive intermediate to form the two major iridoid stereoisomers found in plants. Our work uncovers the last missing key step in the otherwise well-characterized iridoid biosynthesis pathway in asterids. This discovery unlocks the possibility to generate previously inaccessible iridoid stereoisomers, which will enable metabolic engineering for the sustainable production of valuable iridoid and iridoid-derived compounds.

plant biology↗

bioRxiv2025

Single-cell metabolome and RNA-seq multiplexing on single plant cells

Plants produce valuable natural products used for a wide variety of industrial applications. Since these molecules have important applications in a variety of industrial sectors, there is enormous interest in elucidating the biosynthetic pathways that are responsible for the production of these compounds. Identification of the genes that comprise these biosynthetic pathways has been enabled by gene-to-metabolite networks that are generated from transcriptomic and metabolomic datasets. Recent advances in both single-cell RNA-seq (scRNA-seq) and single-cell mass spectrometry metabolomics (scMS) have enabled the measurement of either gene expression or metabolite levels in individual cells. However, these individual datasets can only be used to indirectly correlate gene expression levels with metabolite concentrations at the single cell level. Here we demonstrate that both scRNA-seq and scMS can be applied to the same plant cell, thereby enabling direct comparisons between gene expression and metabolite levels. This multiplexing approach reveals both qualitative and quantitative correlations between metabolite levels and biosynthetic gene expression in individual cells. This integrated approach sheds light on the underlying processes driving complex plant biosynthesis.

plant biology↗

bioRxiv2025

Isotopic Labeling Analysis using Single Cell Mass Spectrometry

Here we show that a recently developed single cell mass spectrometry method can be used to monitor the incorporation of an isotopically labeled precursor into single plant protoplasts. Specifically, we show that we can monitor the incorporation of deuterium-labeled tryptamine into an alkaloid pathway over the course of 24 hours. The resulting data provides a glimpse into the rate of synthesis and transport of chemically complex monoterpene indole alkaloids in single cells across a small population. By measuring the concentration of labeled alkaloids, we gain insight into the flux of an important plant biosynthetic pathway. Stable-isotope labeling is a widely used approach to study metabolic networks by tracking isotopologues over time. This manuscript provides proof of principle that single cell isotopic labeling can be performed in plant pathways.

plant biology↗

bioRxiv2025

It runs in the family: Discovery of enzymes in the oleuropein pathway in Olive (Olea europaea) by comparative transcriptomics

Olive (Olea europaea L.) is one of the most important crop trees, with olive oil being a key ingredient of the Mediterranean diet. Oleuropein, an oleoside-type secoiridoid, is the major determinant of flavor and quality of olive oil. Iridoid biosynthesis has been elucidated in Catharanthus roseus, which produces secologanin-type secoiridoids, but iridoid biosynthesis in other species remains unresolved. In this work, we sequenced RNA from olive fruit mesocarp of six commercial olive cultivars with varying oleuropein content, during maturation and ripening. Using this data we discovered three polyphenol oxidases with oleuropein synthase (OS) activity, a novel oleoside-11-methyl ester glucosyl transferase (OMEGT) synthesizing a potential intermediate in the route, and a 7-epi-loganic acid O-methyltransferase (7eLAMT). Interestingly, integrating transcriptomics data from 15 plant species from three iridoid-producing plant orders (Lamiales, Gentianales, and Cornales), and tissue expression panels from Jasminum sambac and Fraxinus excelsior, we discovered two 2-oxoglutarate dependent dioxygenases (named 7eLAS) that synthesize 7-epi-loganic acid; in contrast C. roseus 7-deoxy-loganic acid hydroxylase (7DLH), a known bottleneck in MIA production, is a cytochrome p450. This comparative co-expression method, which combines guilt by association and comparative transcriptomics approaches, can successfully leverage big datasets for untargeted discovery of enzymes. Key FindingsO_LIExpression of genes involved in iridoid biosynthesis, from the early MEP pathway to the last step of oleuropein biosynthesis, decreases during olive fruit maturation. C_LIO_LIWe discovered an oxoglutarate dependent dioxygenase, 7-epi-loganic acid synthase (7eLAS), catalyzing the stereoselective oxidation of 7-deoxy-loganic acid to 7-epi-loganic acid, in a reaction analogous to C. roseus 7-deoxy-loganic acid hydroxylase (7DLH), a cytochrome p450. C_LIO_LIWe report a 7-epi-loganic acid O-methyltransferase (7eLAMT) orthologous to Catharanthus roseus loganic acid O-methyltransferase and found a novel oleoside-11-methyl ester glucosyl transferase (OMEGT) synthesizing 7-{beta}-1-D-glucopyranosyl-oleoside-11-methyl ester, a potential intermediate in the oleuropein biosynthesis route. C_LIO_LIWe discovered three olive polyphenol oxidases that have oleuropein synthase (OS) activity, catalyzing the conversion of ligstroside to oleuropein. C_LI

biochemistry↗

bioRxiv2025

BAHD news from Euphorbia peplus: identification of acyltransferase enzymes involved in ingenane diterpenoid biosynthesis

O_LIThe plant family Euphorbiaceae are an abundant source of structurally complex diterpenoids, many of which have reported anti-cancer, anti-HIV, and anti-inflammatory activities. Among these, ingenol-3-angelate (1a; tradename: Picato(R)), isolated from Euphorbia peplus, has potent anti-tumour activity. C_LIO_LIHere we report the discovery and characterization of the first genes linked to the committed steps of ingenol-3-angelate (1a) biosynthesis in Euphorbia peplus. We identified two genes, the products of which catalyse the addition of angelyl-CoA (9a) to the ingenol (5) scaffold to produce ingenol-3-angelate (1a). C_LIO_LIWe demonstrate using VIGS that just one of these genes, EpBAHD-08, is essential for this angeloylation in E. peplus. VIGS of the second gene, EpBAHD-06, has a significant effect on jatrophanes rather than ingenanes in E. peplus. C_LIO_LIWe also identified three genes whose products can catalyse acetylation of ingenol-3-angelate (1a) to ingenol-3-angelate-20-acetate (2). In this case VIGS indicates considerable functional redundancy in the E. peplus genome of genes encoding this enzymatic step. C_LIO_LIThis work paves the way for increasing ingenol-3-angelate (1a) levels in planta and provides a foundation for the discovery of the remaining genes in the biosynthetic pathway of these important molecules. C_LI

biochemistry↗

bioRxiv2024

Enzymatic epimerization of monoterpene indole alkaloids in Kratom

Monoterpene indole alkaloids (MIAs) are a large, structurally diverse class of bioactive natural products. These compounds are biosynthetically derived from a stereoselective Pictet-Spengler condensation that generates a tetrahydro-{beta}-carboline scaffold characterized by a 3S stereocenter. However, a subset of MIAs contain a non-canonical 3R stereocenter. Herein, we report the basis for 3R-MIA biosynthesis in Mitragyna speciosa (Kratom). We discover the presence of the iminium species, 20S-3-dehydrocorynantheidine, which led us to hypothesize that isomerization of 3S to 3R occurs by oxidation and stereoselective reduction downstream of the initial Pictet-Spengler condensation. Isotopologue feeding experiments implicated young leaves and stems as the sites for pathway biosynthesis, facilitating the identification of an oxidase/reductase pair that catalyzes this epimerization. This enzyme pair has broad substrate specificity, suggesting that the oxidase and reductase may be responsible for the formation of many 3R-MIAs and downstream spirooxindole alkaloids in Kratom. These enzymes allow biocatalytic access to a range of previously inaccessible pharmacologically active compounds.

biochemistry↗

bioRxiv2024

Single-cell mass spectrometry reveals heterogeneous triterpenic acid accumulation in apple callus-derived cells

The use of plant cell cultures for large scale production of natural compounds, although promising, has been hindered by their genetic instability and heterogeneity. Here, we show how single cell mass spectrometry can be used to characterize the natural product profile of a callus culture at a highly resolved level. We identify and quantify triterpenic acids in a population of callus cells derived from Annurca apple (Malus pumila Miller cv Annurca) leaf. The analysis demonstrated that a high degree of metabolic heterogeneity exists in the cell population, with the levels of detected metabolites varying significantly across the callus cells. This metabolic heterogeneity was underpinned by variable expression levels of key biosynthetic genes in the single cells. The application of an abiotic stress, near ultraviolet radiation (NUV), to the callus culture resulted in increased levels of triterpenic acids. Single cell mass spectrometry analysis revealed that after treatment, a larger percentage of callus cells produced detectable amounts of these metabolites, ultimately resulting in a more homogeneous production of the metabolites. Furthermore, it showed that intracellular concentrations of ursolic acid derivatives can reach more than 100 mM. Single cell mass spectrometry analyses provide a starting foundation for understanding the molecular mechanisms responsible for metabolic heterogeneity in plant cell cultures, which could in turn facilitate efforts to improve these cell cultures for commercial purposes.

plant biology↗

bioRxiv2024

Divergent biosynthesis of monoterpene indole alkaloids from geissoschizine

Plants can generate structural diversity by enzymatic rearrangement of a central intermediate. 19E-geisssochizine is one such chemically versatile intermediate that plays a central role in the biosynthesis of monoterpene indole alkaloids such as strychnine, ibogaine and vinblastine. Here we report how 19E-geissoschizine undergoes oxidative transformations to generate four distinct alkaloid scaffolds through the action of three biosynthetic enzymes. Using in vitro enzymatic assays and gene silencing, we demonstrate how these three cytochrome P450 enzymes in the medicinal plant Catharanthus roseus transform 19E-geisssochzine into strychnos, sarpagan, akuammiline-type, and mavacurane-type alkaloids. We use mutational analysis to show how minimal changes to the active site of these similar enzymes modulate product specificity. This work highlights how substrate reactivity and enzyme mutations work synergistically to generate chemical diversity.

biochemistry↗

bioRxiv2024

Biosynthetic Origin of the Methoxy Group in Quinine and Related Cinchona Alkaloids

Quinine is a historically important natural product containing a methoxy group that is assumed to be incorporated at a late pathway stage. Here we show that the methoxy group in quinine and related Cinchona alkaloids is introduced onto the starting substrate tryptamine. Feeding studies with Cinchona plantlets definitively show that 5-methoxytryptamine is utilized as a quinine biosynthetic intermediate in planta. We discover the biosynthetic genes that encode the responsible oxidase and methyltransferase, and we use these genes to reconstitute the early steps of the Cinchona alkaloid biosynthetic pathway in Nicotiana benthamiana to produce a mixture of methoxylated and desmethoxylated Cinchona alkaloid intermediates. Importantly, we show that the co-occurrence of both tryptamine and 5-methoxytryptamine substrates, along with the substrate promiscuity of downstream pathway enzymes, enable parallel formation of both methoxylated and desmethoxylated alkaloids in Cinchona pubescens.

plant biology↗

bioRxiv2024

Independent evolution of ipecac alkaloid biosynthesis

Ipecac alkaloids are medicinal monoterpenoid-derived tetrahydroisoquinoline alkaloids found in two distantly related plants: Carapichea ipecacuanha (Gentianales) and Alangium salviifolium (Cornales). We have elucidated ipecac alkaloid biosynthesis in both species, conclusively demonstrating that biosynthesis of the structurally complex ipecac alkaloid protoemetine has evolved independently. We show that although protoemetine biosynthesis proceeds via the same chemical logic in both species, each plant uses a distinct monoterpene precursor. Moreover, we provide evidence that both plants initiate ipecac biosynthesis by a non-enzymatic Pictet-Spengler reaction, and we elucidate the biosynthetic fate of both the 1R and 1S stereoisomers that are produced in this non-stereoselective reaction. Phylogenetic analyses clearly show independent pathway evolution through parallel and convergently evolved enzymes. This work provides insight into how nature can capitalize on highly reactive starting substrates, the manner in which multi-step pathways can arise, and also lays the foundation for metabolic engineering of these important medicinal compounds.

plant biology↗

bioRxiv2024

Highjacked by a pseudoenzyme: How eudicot plants make indole

Indole is crucial for plant defense, where it is released as a signaling volatile upon herbivore attack and also serves as a starting precursor for defensive specialized metabolites. Indole is known to be synthesized in plants from indole-3-glycerol phosphate by the enzyme indole-3-glycerol phosphate lyase. Here we report that in core eudicots, indole production for plant defense occurs via an alternative pathway. The subunit of tryptophan synthase (TSA), an enzyme of core metabolism, normally binds to tryptophan synthase {beta} subunit (TSB) to produce tryptophan. However, we show that a non-catalytic TSB paralogue (TSB-like) can highjack TSA to produce indole. The widespread occurrence of TSB-like genes in eudicots suggests that this alternative mechanism for indole formation is widespread throughout the plant kingdom.

plant biology↗

bioRxiv2024

Streamlined screening platforms lead to the discovery of pachysiphine synthase from Tabernanthe iboga

Plant-specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. The monoterpene indole alkaloids tabersonine and pseudo-tabersonine, found in the medicinal plant Tabernanthe iboga, are extensively modified by oxidative reactions. Here we developed a streamlined screening strategy to screen the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo-tabersonine scaffolds, we set out to uncover the CYP450s responsible for oxidative transformations of these scaffolds. Our approach identified two T. iboga cytochrome P450 enzymes: pachysiphine synthase (PS) and 16-hydroxy-tabersonine synthase (T16H). These enzymes catalyze an epoxidation and site-specific hydroxylation of tabersonine to produce pachysiphine and 16-OH-tabersonine, respectively. We further demonstrated that these genes produced the expected products when expressed in Catharanthus roseus flowers. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and C. roseus as platforms for the functional characterization of plant enzymes.

biochemistry↗

bioRxiv2024

Three cytochrome P450 from Nicotiana attenuata play key roles in triterpene biosynthesis

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant, Nicotiana attenuata. Despite their ecological significance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Three multifunctional cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, NaCYP716E107) from N. attenuata were shown to oxidize the pentacyclic triterpene skeleton as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of {beta}-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2 position of {beta}-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6{beta} position of {beta}-amyrin/oleanolic acid. Three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps. One-sentence summaryThree CYP716 enzymes diversify N. attenuatas triterpenoid sector with potential roles in growth and development.

plant biology↗

bioRxiv2024

Cell-type aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anti-cancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. Here, we generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF) binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific transcription factors as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast specific TF (Idioblast MYB1, CrIDM1) that activates expression of late stage vinca alkaloid biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast specific biosynthetic genes within an idioblast metabolic regulon, but also provides insights into cell-type-specific metabolic regulation.

plant biology↗