Marc Clastre

Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus

Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-PCR strategy based on the design of degenerated oligonucleotides. The deduced amino acid sequence of crdxr is homologue to 1-deoxy-D-xylulose 5-phosphate reductoisomerases. Crmecs represents the first plant cDNA encoding a protein similar to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Escherichia coli. Expression of crdxr and crmecs genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

De novo production of the plant-derived alkaloid strictosidine in yeast.

Significance Plants make a wide variety of complex molecules with potent biological activities including several anticancer therapeutics. Unfortunately, plants often produce these molecules in low amounts, making them expensive to obtain. Engineering simpler organisms, such as yeast, to produce these plant-derived compounds provides one solution to production challenges. One group of plant-derived molecules, the monoterpene indole alkaloids, is synthesized from a common intermediate, strictosidine. Here, we report how we developed a yeast strain that produces strictosidine. This required introducing 21 new genes and three gene deletions into the yeast genome. This yeast strain provides an important resource for the production of expensive, complex molecules that plants normally produce in small amounts. The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼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.

Emerging and emerged pathogenic Candida species: beyond the Candida albicans paradigm.

Many ascomycete yeast species from the Candida genus are widely distributed in nature and act as common saprophytic constituents of the normal human microflora. However, some of these fungal species can also become opportunistic pathogens following a transition from a commensal to a pathogenic phase, induced by alterations in the host environment. Candida species thereby rarely trigger infection in healthy people, but take advantage of a locally or systematically impaired immune system to proliferate in the host and cause diseases termed ‘‘candidiasis.’’ Such fungal infections can be subdivided into three major groups: cutaneous (skin and its appendages), mucosal (oropharyngeal, esophageal, and vulvovaginal) and systemic (bloodstream infections, i.e., candidemia and other forms of invasive candidiasis [IC]). While superficial candidiasis (cutaneous and mucosal) is often observed in AIDS patients, oropharyngeal thrush and vaginitis are more frequently seen in immunocompetent infants and adult women, respectively. Candidemia and IC are common in cancer patients or in transplant individuals following immunosuppression. Candidiasis currently represents the fourth leading cause of nosocomial infections, at 8% to 10%, and mortality due to systemic candidiasis remains high, ranging from 15% to 35% depending on the infecting Candida species [1]. Although Candida albicans remains the most frequently isolated agent of candidiasis, non-albicans Candida (NAC) species now account for a substantial part of clinical isolates collected worldwide in hospitals. NAC species of particular clinical importance include Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei (synonym: Issatchenkia orientalis), as well as the less-prominent species Candida guilliermondii, Candida lusitaniae, Candida kefyr, Candida famata (synonym: Debaryomyces hansenii), Candida inconspicua, Candida rugosa, Candida dubliniensis, and Candida norvegensis (Table 1). A complementary set of about 20 opportunistic NAC species is also known, but exhibits lower isolation rates [2].

1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells.

Abstract Terpenoid indole alkaloids (TIAs) arise from the indole and the monoterpene pathways. The latter route derives from isopentenyl diphosphate (IPP). We report on the isolation and characterization of a cDNA ( crdxs ) encoding for 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) in Catharanthus roseus suspension cultures. The enzyme catalyses the formation of the precursor of the non-mevalonate pathway leading to IPP biosynthesis. Expression in Escherichia coli of the truncated DXPS lacking the putative plastid transit peptide revealed that this pseudomature protein was active. crdxs mRNA were detected only in TIA producing-cells and the accumulation of transcripts was found to be associated with TIA production. This result corroborates recent studies obtained with a labelled precursor, which have given evidence that the non-mevalonate pathway is involved in the biosynthesis of the precursor of TIAs secologanin.

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.

The iridoid pathway in Catharanthus roseus alkaloid biosynthesis

The Apocynaceae Catharanthus roseus accumulates a number of monoterpene indole alkaloids (MIAs) that originate from the coupling of the indole and the iridoid pathways. The latter pathway is usually considered as limiting for MIA biosynthesis, but evidence is now strong that the precursors tryptamine (from the indole pathway) and secologanin (from the iridoid pathway) have to be provided within the cells in a concerted manner for sustained MIA synthesis. Secologanin is formed from isopentenyl diphosphate (IPP) in a number of steps, some of which are still unknown. However significant progress has been obtained recently with the characterisation of cDNAs encoding secologanin synthase and the two constituents of geraniol 10-hydroxylase (G10H). IPP itself is formed through both the plastidial methyl-erythritol phosphate (MEP) pathway and the cytosolic mevalonate (MVA) pathway. The MEP pathway comprises 7 steps of which 4 have been identified at the molecular level in C. roseus. This pathway plays a major role in the production of MIAs, but there is now evidence that the MVA pathway serves as a minor source of precursors for iridoid biosynthesis and/or contributes (through protein prenylation) to a fine regulation of the MEP gene expression. G10H is one of the key enzymes of the MIA pathway and the up-regulation of the gene activity concomitantly with an increase in G10H activity and MIA production have been reported with various hormones and elicitors. Since regulatory genes encoding transcription factors acting on several genes of the MEP and terpenoid pathways are beginning to be characterised, metabolic engineering of the iridoid pathway could be a promising approach to control the metabolite flux towards secologanin and MIA production through biotechnological applications in the future.

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.