Nathalie Giglioli-Guivarc’h

Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta

In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.

Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma.

Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a K(m) value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.

A look inside an alkaloid multisite plant: the Catharanthus logistics.

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.

A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ dependent manner in Catharanthus roseus

A newly identified cytochrome P450 isoform initiates the synthesis of valuable alkaloids in leaves of Catharanthus roseus by hydroxylating tabersonine. Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Candida guilliermondii: biotechnological applications, perspectives for biological control, emerging clinical importance and recent advances in genetics

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last 40 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 and the creation of numerous databases dedicated to gene ontology annotation (metabolic pathways, virulence, and morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.

CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathwayin Catharanthus roseus cells

CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common α subunit and a specific β subunit. We have established RNA interference cell lines targeting the β subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

Antifungal Activity of Resveratrol Derivatives against Candida Species

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.

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondiiku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.

Fluorescent protein fusions in Candida guilliermondii

Candida guilliermondii is an emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains a promising biotechnological model for the industrial production of value-added metabolites. In the present study, we developed a recipient strain as well as a set of plasmids for construction of fluorescent protein (FP) fusions in this species. We demonstrated that C. guilliermondii phosphoglycerate kinase transcription-regulating sequences allow a constitutive expression of codon-optimized green, cyan, yellow and mCherry FP genes in C. guilliermondii cells and the fluorescence signal could be directly observed at the colony and blastospore level by epifluorescence microcopy. To illustrate differential targeting of the FPs into specified cellular compartments, we studied and validated the expected subcellular localization of various C. guilliermondii predicted proteins fused to FPs. Furthermore, co-expression experiments of various couples of FP-tagged C. guilliermondii predicted proteins in the same cell showed that the fluorescence of each FP could be detected independently, providing firm evidences that YFP/CFP and GFP/mCherry pairs can be used for dual labeling in C. guilliermondii cells. This technical advance will facilitate future studies of protein co-expression and co-localization in C. guilliermondii and will give precious help for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering.