Classification of grape seed residues from distillation industries in Europe according to the polyphenol composition highlights the influence of variety, geographical origin and color.

Grape seed residues represent the raw material to produce several value-added products including polyphenol-rich extracts with nutritional and health attributes. Although the impact of variety and environmental conditions on the polyphenol composition in fresh berries is recognized, no data are available regarding grape seed residues. The chemical composition of grape seed residues from wine distilleries in France, Spain and Italy was characterized by mass spectrometry. Forty-two metabolites were identified belonging to non-galloylated and galloylated procyanidins as well as amino acids. Polyphenol concentrations in the red varieties originated from Champagne or Veneto were twice higher than in white varieties from the Loire Valley. The chemical profiles of grape seed residues were mainly classified according to the color variety with galloylated procyanidins as biomarkers of white varieties and non-galloylated procyanidins as biomarkers of red ones. The present approach might assist the selection of grape seed residues as quality raw materials for the production of polyphenol-rich extracts.

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.

Grapevine woody tissues accumulate stilbenoids following bud burst.

MAIN CONCLUSION: After bud burst, a transcriptional reprogramming of the shikimate and phenylpropanoid pathways occurs in grapevine canes resulting in the accumulation of stilbenoids like resveratrol and viniferin. Stilbenoids are phenylpropanoid compounds with important biological properties and biotechnological applications that are synthesized in grapevine in response to different stresses. Although they are found in woody tissues, such as canes and buds, their biosynthesis and accumulation have been essentially described in berries. We have previously shown that transcripts encoding secondary metabolism enzymes accumulate in grapevine canes following the transition from dormancy (E-L 1) to bud burst (E-L 4) suggesting that secondary metabolites may accumulate in grapevine canes during this transition. In the present study, using UPLC-MS we demonstrate the accumulation of important metabolites such as ferulic acid and the stilbenoids E-resveratrol, E-piceatannol and E-ε-viniferin. Stilbenoids accumulation correlated with the increased expression of several stilbene synthase genes and of VviMYB14, encoding a transcription factor that regulates stilbene biosynthesis. In addition, a general stimulation of the plastidial shikimate pathway was observed. Taken together, results show that important secondary metabolites accumulate in the woody canes during bud burst. These findings may aid biotechnological approaches aimed at extracting biologically active phenolic compounds, including stilbenoids, from grapevine woody tissues.

Shoot Cultures of Vitis vinifera (Vine Grape) Different Cultivars as a Promising Innovative Cosmetic Raw Material-Phytochemical Profiling, Antioxidant Potential, and Whitening Activity.

The primary purpose of this work was the initiation and optimization of shoot cultures of different Vitis vinifera L. cultivars: cv. Chardonnay, cv. Hibernal, cv. Riesling, cv. Johanniter, cv. Solaris, cv. Cabernet Cortis, and cv. Regent. Cultures were maintained on 30-day growth cycles using two media, Murashige and Skoog (MS) and Schenk and Hildebrandt (SH), with various concentrations of plant growth regulators. Tested media ('W1'-'W4') contained varying concentrations of 6-benzylaminopurine (BA) in addition to indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA). High performance liquid chromatography coupled with mass spectrometry (UPLC-MS) was used for metabolomic profiling. In all tested extracts, 45 compounds were identified (6 amino acids, 4 phenolic acids, 13 flavan-3-ols, 3 flavonols, and 19 stilbenoids). Principal component analysis (PCA) was performed to assess the influence of the genotype and medium on metabolic content. PCA showed that metabolic content was mainly influenced by genotype and to a lesser extent by medium composition. MS media variants induced the amino acid, procyanidin, and flavan-3-ol production. In addition, the antioxidant potential and anti-tyrosinase activity was measured spectrophotometrically. The studies on antioxidant activity clearly reveal very high efficiency in reducing free radicals in the tested extracts. The strongest tyrosinase inhibition capacity was proved for shoots cv. Hibernal cultured in SH medium and supplemented with NAA, with an inhibition of 17.50%. These studies show that in vitro cultures of V. vinifera cvs. can be proposed as an alternative source of plant material that can be potentially used in cosmetic industry.

Combined omics approaches expose metabolite-microbiota correlations in grape berries of three cultivars of Douro wine region.

This study hypothesized the existence of cultivar-associated correlations between grape berry metabolites and its microbial residents, in Douro wine region. Integrated metabolomics with metabarcoding showed that the microbial biodiversity is not associated to berry sugar concentration, but closely connected to the profile of amino acids, flavonoids and wax compounds, which drove cultivar differentiation together with the prevalence of pathogenic fungi, yeasts and bacteria, mainly Dothideomycetes and Gammaproteobacteria. Over 7000 metabolite-microbiota correlations with ρ >|0.99| exposed a core of 15 metabolites linked to 11 microbial taxa. Serine, oxalate, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, gallic acid, germanicol, sitosterol and erythrodiol correlated negatively to the abundance of most taxa, including Alternaria, Aureobasidium, Pseudopithomyces, Pseudomonas and Sphingomonas. In contrast, phenylalanine, asparagine, alanine, (epi)gallocatechin and procyanidin gallate mediated positive metabolite-OTU correlations. E. necator and A. carbonarius correlated negatively with stigmasterol and amyrin. Complex fungi-bacteria relationships ruled by Dothideomycetes and Alphaproteobacteria further suggest tight host-microbe interactions at the carposphere.

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.

Benzothiadiazole Affects Grape Polyphenol Metabolism and Wine Quality in Two Greek Cultivars: Effects during Ripening Period over Two Years.

Grape berries are one of the most important sources of phenolic compounds, either consumed fresh or as wine. A pioneer practice aiming to enrich grape phenolic content has been developed based on the application of biostimulants such as agrochemicals initially designed to induce resistance against plant pathogens. A field experiment was conducted in two growing seasons (2019-2020) to investigate the effect of benzothiadiazole on polyphenol biosynthesis during grape ripening in Mouhtaro (red-colored) and Savvatiano (white-colored) varieties. Grapevines were treated at the stage of veraison with 0.3 mM and 0.6 mM benzothiadiazole. The phenolic content of grapes, as well as the expression level of genes involved in the phenylpropanoid pathway were evaluated and showed an induction of genes specifically engaged in anthocyanins and stilbenoids biosynthesis. Experimental wines deriving from benzothiadiazole-treated grapes exhibited increased amounts of phenolic compounds in both varietal wines, as well as an enhancement in anthocyanin content of Mouhtaro wines. Taken together, benzothiadiazole can be utilized to induce the biosynthesis of secondary metabolites with oenological interest and to improve the quality characteristics of grapes produced under organic conditions.

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.

Genome Assembly of the Medicinal Plant Voacanga thouarsii.

The Apocynaceae tree Voacanga thouarsii, native to southern Africa and Madagascar, produces monoterpene indole alkaloids (MIA), which are specialized metabolites with a wide range of bioactive properties. Voacanga species mainly accumulates tabersonine in seeds making these species valuable medicinal plants currently used for industrial MIA production. Despite their importance, the MIA biosynthesis in Voacanga species remains poorly studied. Here, we report the first genome assembly and annotation of a Voacanga species. The combined assembly of Oxford Nanopore Technologies long-reads and Illumina short-reads resulted in 3,406 scaffolds with a total length of 1,354.26 Mb and an N50 of 3.04 Mb. A total of 33,300 protein-coding genes were predicted and functionally annotated. These genes were then used to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. A transposable element (TE) analysis showed the highest proportion of TE in Voacanga thouarsii compared with all other MIA-producing plants. In a nutshell, this first reference genome of V. thouarsii will thus contribute to strengthen future comparative and evolutionary studies in MIA-producing plants leading to a better understanding of MIA pathway evolution. This will also allow the potential identification of new MIA biosynthetic genes for metabolic engineering purposes.

Abscisic Acid and Chitosan Modulate Polyphenol Metabolism and Berry Qualities in the Domestic White-Colored Cultivar Savvatiano.

During the last decade, several studies demonstrated the effect of biostimulants on the transcriptional and metabolic profile of grape berries, suggesting their application as a useful viticultural practice to improve grape and wine quality. Herein, we investigated the impact of two biostimulants-abscisic acid (0.04% w/v and 0.08% w/v) and chitosan (0.3% w/v and 0.6% w/v)-on the polyphenol metabolism of the Greek grapevine cultivar, Savvatiano, in order to determine the impact of biostimulants' application in the concentration of phenolic compounds. The applications were performed at the veraison stage and the impact on yield, berry quality traits, metabolome and gene expression was examined at three phenological stages (veraison, middle veraison and harvest) during the 2019 and 2020 vintages. Results showed that anthocyanins increased during veraison after treatment with chitosan and abscisic acid. Additionally, stilbenoids were recorded in higher amount following the chitosan and abscisic acid treatments at harvest. Both of the abscisic acid and chitosan applications induced the expression of genes involved in stilbenoids and anthocyanin biosynthesis and resulted in increased accumulation, regardless of the vintage. Alterations in other phenylpropanoid gene expression profiles and phenolic compound concentrations were observed as well. Nevertheless, they were mostly restricted to the first vintage. Therefore, the application of abscisic acid and chitosan on the Greek cultivar Savvatiano showed promising results to induce stilbenoid metabolism and potentially increase grape defense and quality traits.

Identifying Major Drivers of Antioxidant Activities in Complex Polyphenol Mixtures from Grape Canes.

Grape canes represent a valuable source of numerous polyphenols with antioxidant properties, whose compositions vary depending on the genotype and environmental factors. Antioxidant activities of pure molecules are often reported without considering possible interactions that may occur in complex polyphenol mixture. Using UPLC-MS-based metabolomics and unsupervised classification, we explored the polyphenol variations in grape cane extracts from a collection of European varieties. Antioxidant activities were assessed using ORAC, ABTS, DPPH, FRAP, CUPRAC and chelation assays. Pairwise correlations between polyphenols and antioxidant capacities were performed to identify molecules that contributed more to the antioxidant capacities within a complex mixture of polyphenols.

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus.

Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.

Impact of Deficit Irrigation on Grapevine cv. 'Touriga Nacional' during Three Seasons in Douro Region: An Agronomical and Metabolomics Approach.

The introduction of irrigation in vineyards of the Mediterranean basin is a matter of debate, in particular in those of the Douro Demarcated Region (DDR), due to the limited number of available studies. Here, we aimed to perform a robust analysis in three consecutive vintages (2018, 2019, and 2020) on the impact of deficit irrigation on the yield, berry quality traits, and metabolome of cv. 'Touriga Nacional'. Results showed that in the peaks of extreme drought, irrigation at 30% crop evapotranspiration (ETc) (R30) was able to prevent a decay of up to 0.4 MPa of leaf predawn water potential (ΨPd), but irrigation at 70% ETc (R70) did not translate into additional protection against drought stress. Following three seasons of irrigation, the yield was significantly improved in vines irrigated at R30, whereas irrigation at R70 positively affected the yield only in the 2020 season. Berry quality traits at harvest were not significantly changed by irrigation, except for Total Soluble Solids (TSS) in 2018. A UPLC-MS-based targeted metabolomic analysis identified eight classes of compounds, amino acids, phenolic acids, stilbenoid DP1, stilbenoid DP2, flavonols, flavan-3-ols, di-OH- and tri-OH anthocyanins, and showed that anthocyanins and phenolic acids did not change significantly with irrigation. The present study showed that deficit irrigation partially mitigated the severe summer water deficit conditions in the DDR but did not significantly change key metabolites.

Tonoplast and Peroxisome Targeting of γ-tocopherol N-methyltransferase Homologs Involved in the Synthesis of Monoterpene Indole Alkaloids.

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N-methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit NMT activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta, our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis.

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

Faba bean root exudates alter pea root colonization by the oomycete Aphanomyces euteiches at early stages of infection.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

Exogenous Calcium Delays Grape Berry Maturation in the White cv. Loureiro While Increasing Fruit Firmness and Flavonol Content.

Vineyard calcium (Ca) sprays have been increasingly used by grape growers to improve fruit firmness and thus maintain quality, particularly in periods of heavy rains and hail. The observation that Ca visibly modified berry size, texture, and color in the most prominent white cultivar of the DOC region 'Vinhos Verdes', cultivar (cv.) Loureiro, led us to hypothesize that Ca induced metabolic rearrangements that resulted in a substantial delay in fruit maturation. Targeted metabolomics by ultra-performance liquid chromatography coupled to mass spectrometry and directed transcriptomics were thus combined to characterize the metabolic and transcriptional profiles of cv. Loureiro berries that, together with firmness, °Brix, and fruit weight measurements, allowed to obtain an integrated picture of the biochemical and structural effects of Ca in this cultivar. Results showed that exogenous Ca decreased amino acid levels in ripe berries while upregulating PAL1 expression, and stimulated the accumulation of caftaric, coutaric, and fertaric acids. An increase in the levels of specific stilbenoids, namely E-piceid and E-ω-viniferin, was observed, which correlated with the upregulation of STS expression. Trace amounts of anthocyanins were detected in berries of this white cultivar, but Ca treatment further inhibited their accumulation. The increased berry flavonol content upon Ca treatment confirmed that Ca delays the maturation process, which was further supported by an increase in fruit firmness and decrease in weight and °Brix at harvest. This newly reported effect may be specific to white cultivars, a topic that deserves further investigation.

Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments.

Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.