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Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol.

ABSTRACT: Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus's geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.

SUBMITTER: Saiman MZ

PROVIDER: S-EPMC6445406 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol.

Saiman Mohd Zuwairi MZ Miettinen Karel K Mustafa Natali Rianika NR Choi Young Hae YH Verpoorte Robert R Schulte Anna Elisabeth AE

Plant cell, tissue and organ culture 20180224 1

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from pl ...[more]

PMID: 31007320

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Project description:BackgroundClinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASβ subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots.ResultsOverexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαβ hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα.ConclusionThe transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαβ engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression.

Dataset Information

Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol.

Publications

Metabolic alteration of <i>Catharanthus roseus</i> cell suspension cultures overexpressing <i>geraniol synthase</i> in the plastids or cytosol.

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