Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine.

Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.

ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

Emerging trends in production of plant natural products and new-to-nature biopharmaceuticals in yeast.

Natural products represent an inestimable source of valuable compounds for human health. Notably, those produced by plants remain challenging to access due to their low production. Potential shortages of plant-derived biopharmaceuticals caused by climate change or pandemics also regularly tense the market trends. Thus, biotechnological alternatives of supply based on synthetic biology have emerged. These innovative strategies mostly rely on the use of engineered microbial systems for compound synthesis. In this regard, yeasts remain the easiest-tractable eukaryotic models and a convenient chassis for reconstructing whole biosynthetic routes for the heterologous production of plant-derived metabolites. Here, we highlight the recent discoveries dedicated to the bioproduction of new-to-nature compounds in yeasts and provide an overview of emerging strategies for optimising bioproduction.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids.

Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine metabolomics, proteomics, transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives.

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.

Medicinal plants enter the single-cell multi-omics era.

Elucidating biosynthetic pathways of plant specialized metabolites is a tricky but essential task for the biotechnological production of plant drugs. In a new report, Li et al. used a single-cell multi-omics approach to provide an integrative view of the architecture and regulation of anticancer alkaloid routes in Madagascar periwinkle.

Terroir Influence on Polyphenol Metabolism from Grape Canes: A Spatial Metabolomic Study at Parcel Scale.

The composition of bioactive polyphenols from grape canes, an important viticultural byproduct, was shown to be varietal-dependent; however, the influence of soil-related terroir factors remains unexplored. Using spatial metabolomics and correlation-based networks, we investigated how continuous changes in soil features and topography may impact the polyphenol composition in grape canes. Soil properties, topography, and grape cane extracts were analyzed at georeferenced points over 3 consecutive years, followed by UPLC-DAD-MS-based metabolomic analysis targeting 42 metabolites. Principal component analyses on intra-vintage metabolomic data presented a good reproducibility in relation to geographic coordinates. A correlation-driven approach was used to explore the combined influence of soil and topographic variables on metabolomic responses. As a result, a metabolic cluster including flavonoids was correlated with elevation and curvature. Spatial metabolomics driven by correlation-based networks represents a powerful approach to spatialize field-omics data and may serve as new field-phenotyping tool in precision agriculture.

The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids.

Cocaine and hyoscyamine are two tropane alkaloids (TA) from Erythroxylaceae and Solanaceae, respectively. These famous compounds possess anticholinergic properties that can be used to treat neuromuscular disorders. While the hyoscyamine biosynthetic pathway has been fully elucidated allowing its de novo synthesis in yeast, the cocaine pathway remained only partially elucidated. Recently, the Huang research group has completed the cocaine biosynthetic route by characterizing its two missing enzymes. This allowed the whole pathway to be transferring into Nicotiana benthamiana to achieve cocaine production. Here, besides highlighting the impact of this discovery, we discuss how TA biosynthesis evolved via the recruitment of two distinct and convergent pathways in Erythroxylaceae and Solanaceae. Finally, while enriching our knowledge on TA biosynthesis, this diversification of the molecular actors involved in cocaine and hyoscyamine biosynthesis opens perspectives in metabolic engineering by exploring enzyme biochemical plasticity that can ease and shorten TA pathway reconstitution in heterologous organisms.

Gaining access to acetyl-CoA by peroxisomal surface display.

Synthetic biology is constantly making progress for producing compounds on demand. Recently, Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane. This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.

Spatial localization of monoterpenoid indole alkaloids in Rauvolfia tetraphylla by high resolution mass spectrometry imaging.

Monoterpenoid indole alkaloids (MIAs) are a large group of biosynthetic compounds, which have pharmacological properties. One of these MIAs, reserpine, was discovered in the 1950s and has shown properties as an anti-hypertension and anti-microbial agent. Reserpine was found to be produced in various plant species within the genus of Rauvolfia. However, even though its presence is well known, it is still unknown in which tissues Rauvolfia produce reserpine and where the individual steps in the biosynthetic pathway take place. In this study, we explore how matrix assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometry imaging (MSI) can be used in the investigation of a proposed biosynthetic pathway by localizing reserpine and the theoretical intermediates of it. The results show that ions corresponding to intermediates of reserpine were localized in several of the major parts of Rauvolfia tetraphylla when analyzed by MALDI- and DESI-MSI. In stem tissue, reserpine and many of the intermediates were found compartmentalized in the xylem. For most samples, reserpine itself was mainly found in the outer layers of the sample, suggesting it may function as a defense compound. To further confirm the place of the different metabolites in the reserpine biosynthetic pathway, roots and leaves of R. tetraphylla were fed a stable-isotope labelled version of the precursor tryptamine. Subsequently, several of the proposed intermediates were detected in the normal version as well as in the isotope labelled versions, confirming that they were synthesized in planta from tryptamine. In this experiment, a potential novel dimeric MIA was discovered in leaf tissue of R. tetraphylla. The study constitutes to date the most comprehensive spatial mapping of metabolites in the R. tetraphylla plant. In addition, the article also contains new illustrations of the anatomy of R. tetraphylla.

Convergent evolution for antibiotic biosynthesis in bacteria and animals.

Convergent evolution has been described for several metabolic pathways across the kingdoms of life. However, there is hitherto no evidence for such an interkingdom process for antimicrobials. A new report suggests that marine animals have evolved the ability to biosynthesize antimicrobial polyketides, in parallel with bacteria.

Identification of a second 16-hydroxytabersonine-O-methyltransferase suggests an evolutionary relationship between alkaloid and flavonoid metabolisms in Catharanthus roseus.

The medicinal plant Catharanthus roseus biosynthesizes many important drugs for human health, including the anticancer monoterpene indole alkaloids (MIAs) vinblastine and vincristine. Over the past decades, the continuous increase in pharmaceutical demand has prompted several research groups to characterize MIA biosynthetic pathways for considering future metabolic engineering processes of supply. In line with previous work suggesting that diversification can potentially occur at various steps along the vindoline branch, we were here interested in investigating the involvement of distinct isoforms of tabersonine-16-O-methyltransferase (16OMT) which plays a pivotal role in the MIA biosynthetic pathway. By combining homology searches based on the previously characterized 16OMT1, phylogenetic analyses, functional assays in yeast, and biochemical and in planta characterizations, we identified a second isoform of 16OMT, referred to as 16OMT2. 16OMT2 appears to be a multifunctional enzyme working on both MIA and flavonoid substrates, suggesting that a constrained evolution of the enzyme for accommodating the MIA substrate has probably occurred to favor the apparition of 16OMT2 from an ancestral specific flavonoid-O-methyltransferase. Since 16OMT1 and 16OMT2 displays a high sequence identity and similar kinetic parameters for 16-hydroxytabersonine, we postulate that 16OMT1 may result from a later 16OMT2 gene duplication accompanied by a continuous neofunctionalization leading to an almost complete loss of flavonoid O-methyltransferase activity. Overall, these results participate in increasing our knowledge on the evolutionary processes that have likely led to enzyme co-optation for MIA synthesis.

Emerging mechanistic insights into the regulation of specialized metabolism in plants.

Plants biosynthesize a broad range of natural products through specialized and species-specific metabolic pathways that are fuelled by core metabolism, together forming a metabolic network. Specialized metabolites have important roles in development and adaptation to external cues, and they also have invaluable pharmacological properties. A growing body of evidence has highlighted the impact of translational, transcriptional, epigenetic and chromatin-based regulation and evolution of specialized metabolism genes and metabolic networks. Here we review the forefront of this research field and extrapolate to medicinal plants that synthetize rare molecules. We also discuss how this new knowledge could help in improving strategies to produce useful plant-derived pharmaceuticals.