Maite Colinas

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 3 S -stereoselective. Here, we report that the medicinal plant species Pogonopus speciosus (Rubiaceae) accumulates the 3 R epimer vincosidic acid produced by a an ortholog of STR. This ortholog, named here Epi-STR, exclusively produces the 3 R 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.

Iridoids are specialized monoterpenes ancestral to asterid flowering plants ^1,2 that play key roles in defence and are also essential precursors for pharmacologically important alkaloids ^3,4 . The biosynthesis of all iridoids involves the cyclization of the reactive biosynthetic intermediate 8-oxocitronellyl enol. Here, using a variety of approaches including single-nuclei sequencing, we report the discovery of iridoid cyclases from a phylogenetically broad sample of asterid species that synthesize iridoids. We show that these enzymes catalyse formation of 7 S - cis-trans and 7 R - cis-cis nepetalactol, the two major iridoid stereoisomers found in plants. Our work uncovers a key missing step in the otherwise well-characterized early 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.

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.

Ipecac alkaloids are medicinal monoterpenoid-derived tetrahydroisoquinoline alkaloids found in two distantly related plants: Carapichea ipecacuanha (Gentianales) and Alangium salviifolium (Cornales). Here we provide evidence suggesting that both plants initiate ipecac alkaloid biosynthesis through a nonenzymatic Pictet–Spengler reaction and we elucidate the biosynthetic fate of both the 1 R and 1 S stereoisomers that are produced in this nonstereoselective reaction. Although the biosynthesis of the 1 S -derived protoemetine proceeds according to the same chemical logic in both species, each plant uses a distinct monoterpene precursor. Phylogenetic analyses show examples of independent pathway evolution through parallel and convergently evolved enzymes. This work provides insight into how nature can capitalize on highly reactive starting substrates and the manner in which multistep pathways can arise and lays the foundation for metabolic engineering of these important medicinal compounds.

Summary: 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 anticancer 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. 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 TFs 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 MIA 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.

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 1 R and 1 S 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.

Summary: Plant‐specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. Monoterpene indole alkaloids (MIAs) tabersonine and pseudo‐tabersonine, found in the medicinal plant Tabernanthe iboga (commonly known as iboga), are tailored with oxidations, and the enzymes involved remain unknown. Here, we developed a streamlined screening strategy to test the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo‐tabersonine scaffolds, we aimed to uncover the CYP450s responsible for oxidative transformations in 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. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and Catharanthus roseus as platforms for the functional characterization of plant enzymes.

Summary: 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.

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 ( Id ioblast M YB1, 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.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5′-phosphate oxidase3 (PDX3), which regulates vitamin B₆ homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO₂, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO₂, likely due to the requirement of pyridoxal 5′-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B₆ homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.