The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Rodrigo B. Andrade - One of the best experts on this subject based on the ideXlab platform.

Senzhi Zhao - One of the best experts on this subject based on the ideXlab platform.

Ka-yiu San - One of the best experts on this subject based on the ideXlab platform.

  • the expression of 1 deoxy d xylulose synthase and geraniol 10 hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in catharanthus roseus hairy roots
    Metabolic Engineering, 2011
    Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Erik H Hughes, Ryan Peacock, Ka-yiu San
    Abstract:

    The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in Tabersonine by 66% and horhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and Tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in horhammericine by 30%, lochnericine by 27% and Tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

  • Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.
    Biotechnology and bioengineering, 2006
    Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu San
    Abstract:

    Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase α subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase α or α and β subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase α subunit, we observed an increase of 125% in horhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase α and β subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and Tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production. © 2005 Wiley Periodicals, inc.

  • Transient effects of overexpressing anthranilate synthase α and β subunits in Catharanthus roseus hairy roots
    Biotechnology progress, 2005
    Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu San
    Abstract:

    Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase α subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase β subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, horhammericine, and Tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

Ines Carqueijeiro - One of the best experts on this subject based on the ideXlab platform.

  • enhanced bioproduction of anticancer precursor vindoline by yeast cell factories
    Microbial Biotechnology, 2021
    Co-Authors: Natalja Kulagina, Johan-owen De Craene, Audrey Oudin, Ines Carqueijeiro, Gregory Guirimand, Celine Melin, Pamela Lemoscruz, Vladimir Heredia, Konstantinos Koudounas
    Abstract:

    The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting Tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant Tabersonine resources.

  • identifying genes involved in alkaloid biosynthesis in vinca minor through transcriptomics and gene co expression analysis
    Biomolecules, 2020
    Co-Authors: Emily Amor Stander, Konstantinos Koudounas, Pamela Lemos Cruz, Sébastien Besseau, Arnaud Lanoue, Nicolas Papon, Liuda Johana Sepulveda, Thomas Duge De Bernonville, Ines Carqueijeiro, Nathalie Giglioliguivarch
    Abstract:

    The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a Tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of Tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxyTabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

  • two Tabersonine 6 7 epoxidases initiate lochnericine derived alkaloid biosynthesis in catharanthus roseus
    Plant Physiology, 2018
    Co-Authors: Ines Carqueijeiro, Khoa Chung, Sébastien Besseau, Arnaud Lanoue, Audrey Oudin, Thomas Duge De Bernonville, Stephanie Brown, Thuthuy T Dang, Manish Walia, Kevin Billet
    Abstract:

    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.

  • a bahd acyltransferase catalyzing 19 o acetylation of Tabersonine derivatives in roots of catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids
    Plant Journal, 2018
    Co-Authors: Ines Carqueijeiro, Arnaud Lanoue, Thomas Duge De Bernonville, Thuthuy T Dang, Kevin Billet, Christiana N Teijaro, Christian Paetz, Angela Mosquera, Audrey Oudin
    Abstract:

    While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to Tabersonine metabolism have been identified to date, namely Tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named Tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxyTabersonine derivatives from roots, such as minovincinine and horhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of Tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

Jacqueline V. Shanks - One of the best experts on this subject based on the ideXlab platform.

  • the expression of 1 deoxy d xylulose synthase and geraniol 10 hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in catharanthus roseus hairy roots
    Metabolic Engineering, 2011
    Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Erik H Hughes, Ryan Peacock, Ka-yiu San
    Abstract:

    The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in Tabersonine by 66% and horhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and Tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in horhammericine by 30%, lochnericine by 27% and Tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

  • Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.
    Biotechnology and bioengineering, 2006
    Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu San
    Abstract:

    Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase α subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase α or α and β subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase α subunit, we observed an increase of 125% in horhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase α and β subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and Tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production. © 2005 Wiley Periodicals, inc.

  • Transient effects of overexpressing anthranilate synthase α and β subunits in Catharanthus roseus hairy roots
    Biotechnology progress, 2005
    Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu San
    Abstract:

    Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase α subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase β subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, horhammericine, and Tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

  • effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by catharanthus roseus hairy root cultures
    Biotechnology Progress, 1998
    Co-Authors: Sushil K Rijhwani, Jacqueline V. Shanks
    Abstract:

    Late exponential phase hairy root cultures of Catharanthus roseus were elicited with pectinase and jasmonic acid. The effects of elicitor concentration and exposure time on growth and levels of several compounds in the indole alkaloid biosynthetic pathway were monitored. Pectinase decreased the fresh weight to dry weight ratio of the roots, while addition of jasmonic acid had no significant effect. Selective effects on indole alkaloid yields were observed upon addition of elicitors. An increase of 150% in Tabersonine specific yield was observed upon addition of 72 units of pectinase. Transient studies at the same level demonstrated possible catabolism as serpentine, Tabersonine, and lochnericine levels decreased immediately after elicitation. The levels of these compounds recovered back to control levels or were higher than the control levels after some time. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic acid addition caused an increase in the specific yields of ajmalicine (80%), serpentine (60%), lochnericine (150%), and horhammericine (500%) in dosage studies. Tabersonine, the likely precursor of lochnericine and horhammericine, decreased at lower levels of jasmonic acid and then increased with increasing jasmonic acid concentration. Transient studies showed that lochnericine and Tabersonine levels go through a maxima, then decrease back to control levels and reduce below control levels, respectively. The yields of ajmalicine, serpentine, and horhammericine increased continuously after the addition of jasmonic acid. The methods described in this article could generally be used in devising strategies for enhancement in productivity of secondary metabolites and for probing and studying the complex secondary metabolite pathways in plant tissue cultures.