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.

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.

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.

Silencing of Oleuropein ß-Glucosidase Abolishes the Biosynthetic Capacity of Secoiridoids in Olives.

Specialized metabolism is an evolutionary answer that fortifies plants against a wide spectrum of (a) biotic challenges. A plethora of diversified compounds can be found in the plant kingdom and often constitute the basis of human pharmacopeia. Olive trees (Olea europaea) produce an unusual type of secoiridoids known as oleosides with promising pharmaceutical activities. Here, we transiently silenced oleuropein ß-glucosidase (OeGLU), an enzyme engaged in the biosynthetic pathway of secoiridoids in the olive trees. Reduction of OeGLU transcripts resulted in the absence of both upstream and downstream secoiridoids in planta, revealing a regulatory loop mechanism that bypasses the flux of precursor compounds toward the branch of secoiridoid biosynthesis. Our findings highlight that OeGLU could serve as a molecular target to regulate the bioactive secoiridoids in olive oils.

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories.

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids - flavor and quality determinants in olive oil.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

Virus-Induced Gene Silencing in Olive Tree (Oleaceae).

Research on gene functions in non-model tree species is hampered by a number of difficulties such as time-consuming genetic transformation protocols and extended period for the production of healthy transformed offspring, among others. Virus-induced gene silencing (VIGS) is an alternative approach to transiently knock out an endogenous gene of interest (GOI) by the introduction of viral sequences encompassing a fragment of the GOI and to exploit the posttranscriptional gene silencing (PTGS) mechanism of the plant, thus triggering silencing of the GOI. Here we describe the successful application of Tobacco rattle virus (TRV)-mediated VIGS through agroinoculation of olive plantlets. This methodology is expected to serve as a fast tracking and powerful tool enabling researchers from diversified fields to perform functional genomic analyses in the olive tree.

Beyond the semi-synthetic artemisinin: metabolic engineering of plant-derived anti-cancer drugs.

The discovery and supply of plant-derived anti-cancer compounds remain challenging given their low bioavailability and structural complexity. Reconstituting the pathways of these compounds in heterologous hosts is a promising solution; however, requires the complete elucidation of the biosynthetic genes involved and extensive metabolic engineering to optimise enzyme activity and metabolic flux. This review describes the current strategies and recent advancements in the production of these valuable therapeutic compounds, and highlights plant-derived immunomodulators as an emerging class of anti-cancer agents.

Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.