Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

A "Magic Mushroom" Multi-Product Sesquiterpene Synthase.

Psilocybe "magic mushrooms" are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, ß-copaene, δ-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of ß-copaene and δ-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

Transcriptome-based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry.

Blueberries (Vaccinium spp.) are well known for their nutritional quality, and recent work has shown that Vaccinium spp. also produce iridoids, which are specialized metabolites with potent health-promoting benefits. The iridoid glycoside monotropein, which has anti-inflammatory and antinociceptive activities, has been detected in several wild blueberry species but in only a few cultivated highbush blueberry cultivars. How monotropein is produced in blueberry and the genes involved in its biosynthesis remain to be elucidated. Using a monotropein-positive (M+) and monotropein-negative (M-) cultivar of blueberry, we employed transcriptomics and comparative genomics to identify candidate genes in the blueberry iridoid biosynthetic pathway. Orthology analysis was completed using de novo transcript assemblies for both the M+ and M- blueberry cultivars along with the known iridoid-producing plant species Catharanthus roseus to identify putative genes involved in key steps in the early iridoid biosynthetic pathway. From the identified orthologs, we functionally characterized iridoid synthase (ISY), a key enzyme involved in formation of the iridoid scaffold, from both the M+ and M- cultivars. Detection of nepetalactol suggests that ISY from both the M+ and M- cultivars produce functional enzymes that catalyze the formation of iridoids. Transcript accumulation of the putative ISY gene did not correlate with monotropein production, suggesting other genes in the monotropein biosynthetic pathway may be more directly responsible for differential accumulation of the metabolite in blueberry. Mutual rank analysis revealed that ISY is co-expressed with UDP-glucuronosyltransferase, which encodes an enzyme downstream of the ISY step. Results from this study contribute new knowledge in our understanding of iridoid biosynthesis in blueberry and could lead to development of new cultivars with increased human health benefits.

Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase.

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Metabolite and Molecular Characterization of Mitragyna speciosa Identifies Developmental and Genotypic Effects on Monoterpene Indole and Oxindole Alkaloid Composition.

The monoterpene indole alkaloid (MIA) mitragynine has garnered attention as a potential treatment for pain, opioid use disorder, and opioid withdrawal because of its combined pharmacology at opioid and adrenergic receptors in humans. This alkaloid is unique to Mitragyna speciosa (kratom), which accumulates over 50 MIAs and oxindole alkaloids in its leaves. Quantification of 10 targeted alkaloids from several tissue types and cultivars of  M. speciosa revealed that mitragynine accumulation was highest in leaves, followed by stipules and stems, but was absent, along with other alkaloids, in roots. While mitragynine is the predominant alkaloid in mature leaves, juvenile leaves accumulate higher amounts of corynantheidine and speciociliatine. Interestingly, corynantheidine has an inverse relationship with mitragynine accumulation throughout leaf development. Characterization of various cultivars of M. speciosa indicated altered alkaloidal profiles ranging from undetectable to high levels of mitragynine. DNA barcoding and phylogenetic analysis using ribosomal ITS sequences revealed polymorphisms leading M. speciosa cultivars having lower mitragynine content to group with other mitragyna species, suggesting interspecific hybridization events. Root transcriptome analysis of low- and high-mitragynine-producing cultivars indicated significant differences in gene expression and revealed allelic variation, further supporting that hybridization events may have impacted the alkaloid profile of M. speciosa.

Directed Biosynthesis of Mitragynine Stereoisomers.

Mitragyna speciosa ("kratom") is used as a natural remedy for pain and management of opioid dependence. The pharmacological properties of kratom have been linked to a complex mixture of monoterpene indole alkaloids, most notably mitragynine. Here, we report the central biosynthetic steps responsible for the scaffold formation of mitragynine and related corynanthe-type alkaloids. We illuminate the mechanistic basis by which the key stereogenic center of this scaffold is formed. These discoveries were leveraged for the enzymatic production of mitragynine, the C-20 epimer speciogynine, and fluorinated analogues.

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana.

Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the late-stage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N. benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.

Recycling Upstream Redox Enzymes Expands the Regioselectivity of Cycloaddition in Pseudo-Aspidosperma Alkaloid Biosynthesis.

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudoaspidosperma alkaloids pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.

In vivo characterization of key iridoid biosynthesis pathway genes in catnip (Nepeta cataria).

MAIN CONCLUSION: Using virus-induced gene silencing, we demonstrated that the enzymes GES, ISY, and MLPL are responsible for nepetalactone biosynthesis in Nepeta cataria. Nepetalactone is the main iridoid that is found in the Nepeta genus and is well-known for its psychoactive effect on house cats. Moreover, there is a burgeoning interest into the effect of nepetalactone on insects. Although the enzymes for nepetalactone biosynthesis have been biochemically assayed in vitro, validation of the role that these enzymes have in planta has not been demonstrated. Virus-induced gene silencing (VIGS) is a silencing method that relies on transient transformation and is an approach that has been particularly successful when applied to a variety of non-model plants. Here, we use a recently designed visual-marker dependent VIGS system to demonstrate that the nepetalactone biosynthetic enzymes GES, ISY, and MLPL impact nepetalactone biosynthesis in Nepeta cataria.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism.

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,ß-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.