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Metabolic engineering (ME) aims to develop efficient microbial cell factories that can produce a wide variety of valuable compounds, ideally at the highest yield and from various feedstocks. We summarize recent developments in ME methods for tailoring different yeast cell factories (YCFs). In particular, we highlight the most timely and cutting-edge molecular tools and strategies for biosynthetic pathway optimization (including genome-editing tools), combinatorial transcriptional and post-transcriptional engineering (cis/trans regulators), dynamic control of metabolic fluxes (e.g., rewiring of primary metabolism), and spatial reconfiguration of metabolic pathways. Finally, we discuss challenges and perspectives for adaptive laboratory evolution (ALE) of yeast to advance ME of microbial cell factories.

International audience; Grape canes are viticulture byproducts representing a sustainable source of valuable bioactive polyphenols. However, varietal origin and postharvest treatment greatly influence the effective concentration of biomolecules, thus limiting industrial development. With the aim to develop grape cane extracts with high polyphenol contents, the selection of a specific grape variety combined with optimized postharvest treatment is the major determinant. A previously described postharvest treatment comprising cutting grapevine stalks of 0.5 cm length and storage at 15–20 °C over 2 weeks was applied on a selection of 44 grape varieties representative of the genetic diversity of the whole European collection and performing the screening of polyphenol contents. Varietal rankings according to major polyphenols (catechin, epicatechin, E-resveratrol, E-piceatannol, E-ϵ-viniferin, E-miyabenol C, ampelopsin A, E-vitisin B, hopeaphenol, and isophopeaphenol) were performed with and without the postharvest treatment. We observed that postharvest treatment greatly influenced the total polyphenol composition but also the ranking of polyphenol-rich varieties. This polyphenol screening of grape canes from a large collection of European varieties revealed the importance of postharvest treatment together with the selection of varieties to develop natural extracts based on grapevine wood biomass enriched with molecules with health benefits.

This work was supported by EU Horizon 2020 research and innovation program [MIAMi project, grant number 814645; MKJ, SEO, VC]; ARD CVL Biopharmaceutical program of the Région Centre-Val de Loire [ETOPOCentre project, VC]; and ANR [project MIACYC – ANR-20-CE43-0010, VC].; International audience; 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

While fungi are widely occupying nature, many species are responsible for devastating mycosis in humans. Such niche diversity explains how quick fungal adaptation is necessary to endow the capacity of withstanding fluctuating environments and to cope with host-imposed conditions. Among all the molecular mechanisms evolved by fungi, the most studied one is the activation of the phosphorelay signalling pathways, of which the high osmolarity glycerol (HOG) pathway constitutes one of the key molecular apparatus underpinning fungal adaptation and virulence. In this review, we summarize the seminal knowledge of the HOG pathway with its more recent developments. We specifically described the HOG-mediated stress adaptation, with a particular focus on osmotic and oxidative stress, and point out some lags in our understanding of its involvement in the virulence of pathogenic species including, the medically important fungi

International audience; Fluorescent proteins (FPs) have become indispensable tools in biological research for labeling cells and proteins and sensing their biochemical activity. By introducing 'folding mutations', Campbell et al. engineered a new GFP variant with dramatically enhanced cellular brightness and stability, facilitating advanced cellular bioimaging applications in neuroscience and beyond.

Cytokinins (CKs) are primarily known as a prominent type of plant hormones with pleiotropic functions such as the control of the cell division and morphogenesis. CKs are also well known to orchestrate plant responses to many types of environmental stresses. More specifically, CKs were previously shown to negatively regulate the response to salinity stress. However, the molecular mechanisms underlying this physiological process have not been investigated in detail. In a new report, Yan and colleagues show that salt stress interrupts the CK transduction pathway by promoting the degradation of some CK signaling modules. This represents an unprecedented advancement in our comprehension of how plants are able to inhibit their own development under stress conditions by interfering with the cell signaling circuitry of a growth hormone.

The marine environment is a huge reservoir of biodiversity and represents an excellent source of chemical compounds, some of which have large economical values. In the urgent quest for new pharmaceuticals, marine-based drug discovery has progressed significantly over the past several decades and we now benefit from a series of approved marine natural products (MNPs) to treat cancer and pain while an additional collection of promising leads are in clinical trials. However, the discovery and supply of MNPs had always been challenging given their low bioavailability and structural complexity. Their manufacture for pre-clinical and clinical development but also commercialization mainly relies upon marine source extraction and chemical synthesis, which are associated with high costs, unsustainability and severe environmental problems. In this review, we discuss how metabolic engineering now raises reasonable expectations for the implementation of microbial cell factories, which may provide a sustainable approach for MNP-based drug supply in the near future.

Horizontal gene transfer (HGT) is a well-documented evolutionary driving phenomenon in prokaryotes and eukaryotes, but its impact on the plant kingdom has remained elusive. A recent study provides compelling evidences, which support the idea that a plant-derived gene allows for the detoxification of plant defense metabolites in a polyphagous arthropod herbivore.

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.

International audience; 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.

International audience

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants., An improved virus-induced gene silencing approach led to the discovery of the alkaloid biosynthetic enzyme serpentine synthase.

International audience; Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by β-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine β-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks β-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of β-glucosidase multimerization, an organization common to many defensive GH1 members.

The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi-synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

Colchicine is among the oldest plant natural products (NPs) still used for treating a broad spectrum of human diseases including gout and other articular inflammation disorders. This molecule is synthesized by several herbaceous species related to the Liliaceae family, but in very low quantities in whole plants. As for many pharmaceutical compounds from plants, the production of colchicine still depends on the natural resource from which it is extracted. From the past decade, metabolic engineering has progressively become a credible alternative for the cost-effective large-scale production of several valuable NPs. In the same vein, Nett and colleagues recently reported an unprecedented advance in the field for colchicine. By using a combination of transcriptomics, metabolomics and pathway reconstitution, Sattely's group deciphered a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes. Besides constituting a benchmark for the elucidation of natural product biosynthetic pathways, it opens unprecedented perspectives regarding metabolic engineering of colchicine biosynthesis.

Grape canes are under-valued byproducts of viticulture that accumulate large amounts of stilbenoids. These specialized metabolites are phytoalexins of Vitis vinifera that play a keyrole in defence of Vitis vinifera against pathogens. Since in vitro assays demonstrated the antifungal activity of several pure stilbenoids, grape cane extracts (GCE) have been proposed as eco-friendly alternative to classical fungicides in a context of sustainable viticulture. Following the grape pruning during winter, a storage period associated to an efficient extraction procedure, are determinant to obtain GCE with optimal stilbenoid titration. Stilbenoid-enriched extracts have shown promising protection in vitro against various grape pathogens including downy mildew, powdery mildew, gray mold and wood diseases. Additionally, large-scale studies in vineyards reported a significant partial protection against downy mildew. Protection level is relative to the concentration of stilbenoids contained in the grape cane extract. Therefore, the huge varietal diversity of grape could be explored to select high-producing varieties for future use as biocontrol agent.

Highlights • Plant specialised metabolites constitute an enormous reservoir of anticancer compounds. • Their low bioavailability in planta fails to reach the demand. • Heterologous pathway reconstitution is a promising alternative to massively producing these compounds. 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.

For decades, plants have represented an inexhaustible source of natural products used in various sectors such as health and industry. However, one recurring problem is the low accumulation of these compounds in planta and, therefore, their production costs and supply. In recent years, unprecedented hope has been brought by the metabolic engineering of microorganisms, which opens up prospects for supply of these molecules at lower cost with high added value. However, many of these productions remained at a laboratory scale. In a recent article published in Nature Communication, Vincent J. J. Martin's team has developed an optimized yeast strain capable of synthesizing not only a huge amount of (S)-reticuline, a major precursor of the plant tetrahydroisoquinoline alkaloid series, but also a whole range of new-to-nature compounds from this prominent family of natural products. This synthesis, reaching industrial scales, thus paves the way to efficient production in microbial cell factories.

Efficiency is a key organizing principle in modern natural product synthesis. Practical criteria include time, cost, and effort expended to synthesize the target, which tracks with step-count and scale. The execution of a natural product synthesis, that is, the sum and identity of each reaction employed therein, falls along a continuum of chemical (abiotic) synthesis on one extreme, followed by the hybrid chemoenzymatic approach, and ultimately biological (biosynthesis) on the other, acknowledging the first synthesis belongs to Nature. Starting materials also span a continuum of structural complexity approaching the target with constituent elements on one extreme, followed by petroleum-derived and "chiral pool" building blocks, and complex natural products (i.e., semisynthesis) on the other. Herein, we detail our approach toward realizing the first synthesis of (-)-melodinine K, a complex bis-indole alkaloid. The total syntheses of monomers (-)-tabersonine and (-)-16-methoxytabersonine employing our domino Michael/Mannich annulation is described. Isolation of (-)-tabersonine from Voacanga africana and strategic biotransformation with tabersonine 16-hydroxylase for site-specific C-H oxidation enabled a scalable route. The Polonovski-Potier reaction was employed in biomimetic fragment coupling. Subsequent manipulations delivered the target. We conclude with a discussion of efficiency in natural products synthesis and how chemical and biological technologies define the synthetic frontier.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

Virus-induced gene silencing (VIGS) is a fast and efficient tool to investigate gene function in plant as an alternative to knock down/out transgenic lines, especially in plant species difficult to transform and challenging to regenerate such as perennial woody plants. In apple tree, a VIGS vector has been previously developed based on the Apple latent spherical virus (ALSV) and an efficient inoculation method has been optimized using biolistics. This report described detailed step-by-step procedure to design and silence a gene of interest (GOI) in apple tree tissues using the ALSV-based vector.

This article belongs to the Special Issue Biosynthesis and Function of Plant Specialized Metabolites., The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis., We acknowledge funding from ARD2020 Biopharmaceutical program of the Région Centre Val de Loire (BioPROPHARM and CatharSIS projects). G.G. acknowledges a research fellowship of Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France.

Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs.

Elucidating plant-specialized biosynthetic pathways has always constituted a laborious task, notably for natural products with high pharmaceutical values. Here, we discuss emerging omics-based strategies that facilitate the identification of genes from these complex metabolic pathways, paving the way to engineered supplies of these compounds through synthetic biology approaches.

International audience; Iridoids are monoterpenes that are produced by various plants as chemical defense molecules. Lichman et al. recently described the timeline of molecular events that underpin the re-emergence of iridoid biosynthesis in an independent lineage of aromatic plants (catnip). This study represents a benchmark for studying enzyme and metabolite evolution in different clades across the tree of life.

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors, histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins, and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus.

International audience; Cyclization reactions that create complex polycyclic scaffolds are hallmarks of alkaloid biosynthetic pathways. We present the discovery of three homologous cytochrome P450s from three monoterpene indole alkaloid-producing plants (Rauwolfia serpentina, Gelsemium sempervirens and Catharanthus roseus) that provide entry into two distinct alkaloid classes, the sarpagans and the β-carbolines. Our results highlight how a common enzymatic mechanism, guided by related but structurally distinct substrates, leads to either cyclization or aromatization.

International audience; No abstract available

Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species.

Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism—an alcohol dehydrogenase and a cytochrome P450—produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products., The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.

BACKGROUND Plasmopara viticola control in organic viticulture requires copper-based fungicides with harmful effects on health and the environment. Plant extracts represent a biorational eco-friendly alternative to copper. The aim of this study was to evaluate the potential of stilbenoid-rich grape cane extract (GCE) against downy mildew on three cultivars over 3 years following natural downy mildew infection. RESULTS Over all field trials, GCE treatments showed an average reduction in disease incidence of -35% and -38% on leaves and clusters, respectively. The average reduction in disease severity was -35% and -43% on leaves and clusters, respectively. Under artificial downy mildew infection, GCE efficacy corresponded to 1 g L-1 of copper. Neither phytotoxicity nor adverse effects on auxiliary fauna were observed after treatment with GCE. CONCLUSION Because few or no biocontrol agents are active alone against P. viticola, GCE is a promising alternative to copper-based fungicides. Grape canes, an abundant by-product of viticulture, have great potential for valorization as a biocontrol agent for sustainable viticulture. © 2018 Society of Chemical Industry.

International audience; Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to horhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhorhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

How to make bioactive alkaloids Vinblastine and vincristine are important, expensive anticancer agents that are produced by dimerization of the plant-derived alkaloids catharanthine and vindoline. The enzymes that transform tabersonine into vindoline are known; however, the mechanism by which the scaffolds of catharanthine and tabersonine are generated has been a mystery. Caputi et al. now describe the biosynthetic genes and corresponding enzymes responsible. This resolves a long-standing question of how plant alkaloid scaffolds are synthesized, which is important not only for vinblastine and vincristine biosynthesis, but also for understanding the many other biologically active alkaloids found throughout nature. Science , this issue p. 1235

Among the subdivision of Saccharomycotina (ascomycetes budding yeasts), the CTG clade (formerly the Candida clade) includes species that display a particular genetic code. In these yeasts, the CTG codon is predominantly translated as a serine instead of a leucine residue. It is now well-known that some CTG clade species have a major impact on human and its activities. Some of them are recognized as opportunistic agents of fungal infections termed candidiasis. In addition, another series of species belonging to the CTG clade draws the attention of some research groups because they exhibit a strong potential in various areas of biotechnology such as biological control, bioremediation, but also in the production of valuable biocompounds (biofuel, vitamins, sweeteners, industrial enzymes). Here we provide an overview of recent advances concerning the biology, clinical relevance, and currently tested biotechnological applications of species of the CTG clade. Future directions for scientific research on these particular yeasts are also discussed.

International audience; In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

International audience; Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Summary The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway., Graphical Abstract, Highlights • Tetrahydroalstonine synthase catalyzes the formation of a plant-derived alkaloid • Tetrahydroalstonine synthase is localized to the nucleus • Tetrahydroalstonine synthase and the preceding pathway enzyme interact • Discovery of a gene controlling structural diversity of monoterpene indole alkaloids, How plants transform the central biosynthetic intermediate strictosidine into thousands of divergent alkaloids has remained unresolved. Stavrinides et al. discover a nuclear-localized alcohol dehydrogenase homolog responsible for conversion of strictosidine aglycone to tetrahydroalstonine that appears to interact with an upstream pathway enzyme.

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that include the anti-cancer agent vincristine, anti-malarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized Nitrate/Peptide Family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold., The stereochemistry of the plant heteroyohimbine alkaloids is a key factor determining their diverse biological activities. Here, the authors carry out structural, localization and genetic experiments to understand the mechanism of stereoselectivity for three heteroyohimbine synthases and to identify their function in vivo.

Phenolamides, so called hydroxycinnamic acid amides, are specialized metabolites produced in higher plants, involved in development, reproduction and serve as defence compounds in biotic interactions. Among them, trihydroxycinnamoyl spermidine derivatives were initially found to be synthetized by a spermidine hydroxycinnamoyltransferase (AtSHT) in Arabidopsis thaliana and to accumulate in the pollen coat. This study reports the identification, in Malus domestica, of an acyltransferase able to complement the sht mutant of Arabidopsis. The quantitative RT-PCR expression profile of MdSHT reveals a specific expression in flowers coordinated with anther development and tapetum cell activities. Three phenolamides including N (1),N (5),N (10)-tricoumaroyl spermidine and N (1),N (5)-dicoumaroyl-N (10)-caffeoyl spermidine identified by LC/MS, were shown to accumulate specifically in pollen grain coat of apple tree. Moreover, in vitro biochemical characterization confirmed MdSHT capacity to synthesize tri-substituted spermidine derivatives with a substrate specificity restricted to p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor. Further investigations of the presence of tri-substituted hydroxycinnamoyl spermidine conjugates in higher plants were performed by targeted metabolic analyses in pollens coupled with bioinformatic analyses of putative SHT orthologues in a wide range of available plant genomes. This work highlights a probable early evolutionary appearance in the common ancestral core Eudicotyledons of a novel enzyme from the BAHD acyltransferase superfamily, dedicated to the synthesis of trihydroxycinnamoyl spermidines in pollen coat. This pathway was maintained in most species; however, recent evolutionary divergences have appeared among Eudicotyledons, such as an organ reallocation of SHT gene expression in Fabales and a loss of SHT in Malvales and Cucurbitales.

International audience; The Madagascar periwinkle produces a large palette of Monoterpenoid Indole Alkaloids (MIAs), a class of complex alkaloids including some of the most valuable plant natural products with precious therapeutical values. Evolutionary pressure on one of the hotspots of biodiversity has obviously turned this endemic Malagasy plant into an innovative alkaloid engine. Catharanthus is a unique taxon producing vinblastine and vincristine, heterodimeric MIAs with complex stereochemistry, and also manufactures more that 100 different MIAs, some shared with the Apocynaceae, Loganiaceae and Rubiaceae members. For over 60 years, the quest for these powerful anticancer drugs has inspired biologists, chemists, and pharmacists to unravel the chemistry, biochemistry, therapeutic activity, cell and molecular biology of Catharanthus roseus. Recently, the ‘‘omics’’ technologies have fuelled rapid progress in deciphering the last secret of strictosidine biosynthesis, the central precursor opening biosynthetic routes to several thousand MIA compounds. Dedicated C. roseus transcriptome, proteome and metabolome databases, comprising organ-, tissue- and cell-specific libraries, and other phytogenomic resources, were developed for instance by PhytoMetaSyn, Medicinal Plant Genomic Resources and SmartCell consortium. Tissue specific library screening, orthology comparison in species with or without MIA-biochemical engines, clustering of gene expression profiles together with various functional validation strategies, largely contributed to enrich the toolbox for plant synthetic biology and metabolic engineering of MIA biosynthesis.

Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine–eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related.

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are similar to 3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.

Grape canes are vineyard waste products containing valuable phytochemicals of medicine and agriculture interest. Grape canes storage is critical for the accumulation of these bioactive compounds. In the present study, we investigated the changes in stilbenoid phytochemical composition during grape cane storage and the influence of the temperature on final concentrations. A strong increase in the concentration of the monomer E-resveratrol (approximately 40-fold) was observed during the first 6 weeks of storage at 20 °C in eight different grape varieties without any change in oligomer concentrations. The E-resveratrol accumulation was temperature-dependent with an optimal range at 15-20 °C. A 2 h heat-shock treatment aiming at protein denaturation inhibited E-resveratrol accumulation. The constitutive expression of key genes involved in the stilbene precursor biosynthesis along with an induction of stilbene synthase (STS) expression during the first weeks of storage contribute to a de novo biosynthesis of E-resveratrol in pruned wood grapes.

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last forty years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260. In this chapter, we provide some recent protocols allowing culture, genetic transformation, gene disruption and fusion to fluorescent protein genes for subcellular localization in this yeast.

Environmental pressures forced plants to diversify specialized metabolisms to accumulate noxious molecules such as alkaloids constituting one of the largest classes of defense metabolites. Catharanthus roseus produces monoterpene indole alkaloids via a highly elaborated biosynthetic pathway whose characterization greatly progressed with the recent expansion of transcriptomic resources. The complex architecture of this pathway, sequentially distributed in at least four cell types and further compartmentalized into several organelles, involves partially identified inter-cellular and intra-cellular translocation events acting as potential key-regulators of metabolic fluxes. The description of this spatial organization and the inherent secretion and sequestration of metabolites not only provide new insight into alkaloid cell biology and its involvement in plant defense processes but also present new biotechnological challenges for synthetic biology.

International audience; trans-Resveratrol (1a) is a phytoalexin produced by plants in response to infections by pathogens. Its potential activity against clinically relevant opportunistic fungal pathogens has previously been poorly investigated. Evaluated herein are the candidacidal activities of oligomers (2a, 3−5) of 1a purified from Vitis vinifera grape canes and several analogues (1b−1j) of 1a obtained through semisynthesis using methylation and acetylation. Moreover, trans-ε-viniferin (2a), a dimer of 1a, was also subjected to methylation (2b) and acetylation (2c) under nonselective conditions. Neither the natural oligomers of 1a (2a, 3−5) nor the derivatives of 2a were active against Candida albicans SC5314. However, the dimethoxy resveratrol derivatives 1d and 1e exhibited antifungal activity against C. albicans with minimum inhibitory concentration (MIC) values of 29−37 μg/mL and against 11 other Candida species. Compound 1e inhibited the yeast-to-hyphae morphogenetic transition of C. albicans at 14 μg/mL. trans Resveratrol (1a) is a natural stilbenoid produced by several plants in response to pathogenic infections. Although present in a restricted number of plant families, 1a forms the backbone of a wide range of stilbene biosynthetic products, leading to a spectrum of monomeric and oligomeric compounds with diverse biological activities.1,2 Stilbenes were first described for their fungicidal action against plant fungal diseases by Langcake and Pryce.3 Later, antimicrobial activities of stilbenes were reported against various phytopathogens, with 1a occurring as a major phytoalexin of the family Vitaceae.4,5 Resveratrol fully inhibits conidial germination of the gray mold agent Botrytis cinerea6 and reduces roughly 75% sporangial germination of the causal agent of grapevine downy mildew, Plasmopara viticola.7 Pterostilbene, the 3,5-dimethoxyresveratrol (1e), and trans ε-viniferin (2a) have been found to be 5- fold more active than 1a, indicating the potential of resveratrol derivatives as a source of effective antifungal agents.8,9 In the field of human health, several investigations have been conducted on 1a due to its multiple pharmacological activities including cardioprotective, antiaging, anticarcinogenic, antiinflammatory, estrogenic/antiestrogenic