Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

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.

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

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 includes the anticancer agent vincristine, antimalarial 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.

Class II Cytochrome P450 Reductase Governs the Biosynthesis of Alkaloids.

Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.

Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.

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.

Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis.

In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome.

BACKGROUND: Transcriptome sequencing offers a great resource for the study of non-model plants such as Catharanthus roseus, which produces valuable monoterpenoid indole alkaloids (MIAs) via a complex biosynthetic pathway whose characterization is still undergoing. Transcriptome databases dedicated to this plant were recently developed by several consortia to uncover new biosynthetic genes. However, the identification of missing steps in MIA biosynthesis based on these large datasets may be limited by the erroneous assembly of close transcripts and isoforms, even with the multiple available transcriptomes. RESULTS: Secologanin synthases (SLS) are P450 enzymes that catalyze an unusual ring-opening reaction of loganin in the biosynthesis of the MIA precursor secologanin. We report here the identification and characterization in C. roseus of a new isoform of SLS, SLS2, sharing 97 % nucleotide sequence identity with the previously characterized SLS1. We also discovered that both isoforms further oxidize secologanin into secoxyloganin. SLS2 had however a different expression profile, being the major isoform in aerial organs that constitute the main site of MIA accumulation. Unfortunately, we were unable to find a current C. roseus transcriptome database containing simultaneously well reconstructed sequences of SLS isoforms and accurate expression levels. After a pair of close mRNA encoding tabersonine 16-hydroxylase (T16H1 and T16H2), this is the second example of improperly assembled transcripts from the MIA pathway in the public transcriptome databases. To construct a more complete transcriptome resource for C. roseus, we re-processed previously published transcriptome data by combining new single assemblies. Care was particularly taken during clustering and filtering steps to remove redundant contigs but not transcripts encoding potential isoforms by monitoring quality reconstruction of MIA genes and specific SLS and T16H isoforms. The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements. CONCLUSIONS: The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway. Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

Insights into B-type RR members as signaling partners acting downstream of HPt partners of HK1 in the osmotic stress response in Populus.

The B-type response regulators (B-type RRs), final elements of a signaling pathway called "histidine/aspartate phosphorelay system" in plants, are devoted to the regulation of response genes through a transcription factor activity. Signal transduction consists in the transfer of a phosphoryl group from a transmembrane histidine kinase (HK) which recognizes a given stimulus to nuclear RRs via cytosolic shuttle phosphotransfer proteins (HPts). In Arabidopsis, the receptors HK are to date the major characterized candidates to be responsible for initiation of osmotic stress responses. However, little information is available concerning the signaling partners acting downstream of HKs. In Populus, three HPts and five B-type RRs were previously identified as interacting partners of HK1, the Arabidopsis AHK1 homolog. Here, we report the isolation of RR18, a member of the B-type RR family, which shares high sequence similarities with ARR18 characterized to act in the osmosensing signaling pathway in Arabidopsis, from poplar cuttings subjected to osmotic stress conditions. By using yeast and in planta interaction assays, RR18 was further identified as acting downstream of HK1 and its three preferential HPt partners. Besides, our results are in favor of a possible involvement of both RR18 and RR13, the main expressed poplar B-type RR, in the osmotic signaling pathway. Nonetheless, different behaviors of these two B-type RRs in this pathway need to be noted, with one RR, RR13, acting in an early phase, mainly in roots of poplar cuttings, and the other one, RR18, acting in a late phase, mainly in leaves to supply an adequate response.

Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids.

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.

Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channeling in secondary metabolism.

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.

Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter-selectable marker.

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii.

A new series of vectors for constitutive, inducible or repressible gene expression in Candida guilliermondii.

The biotechnological potential of C. guilliermondii is now well established. This yeast species currently benefits from the availability of a convenient molecular toolbox including recipient strains, selectable markers and optimized transformation protocols. However, the number of expression systems for biotechnological applications in C. guilliermondii remains limited. We have therefore developed and characterized a new series of versatile controllable expression vectors for this yeast. While previous studies firmly demonstrated that knock-out systems represent efficient genetic strategies to interrupt yeast biochemical pathways at a specific step in C. guilliermondii, the set of expression plasmids described in this study will provide new powerful opportunities to boost homologous or heterologous biosynthetic routes by fine controlled over-expression approaches.

Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii.

Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species.

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

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.

Characterization of an autonomously replicating sequence in Candida guilliermondii.

Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs.

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. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.

Transformation of Candida guilliermondii wild-type strains using the Staphylococcus aureus MRSA 252 ble gene as a phleomycin-resistant marker.

We designed an efficient transformation system for Candida guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C. guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C. guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest.

Optimization of the URA-blaster disruption system in Candida guilliermondii: efficient gene targeting using the URA3 marker.

We established a simple transformation system for C. guilliermondii by developing both an ura3 ATCC 6260-derived recipient strain as well as an URA3 blaster cassette. We demonstrated that this strategy allows efficient multiple gene disruption by homologous recombination with a convenient gene targeting frequency.

A TRP5/5-fluoroanthranilic acid counter-selection system for gene disruption in Candida guilliermondii.

Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3'UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast "TRP blaster" disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created "TRP5 blaster" disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.