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The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus
Saturday, 2015/07/04 | 04:46:30

Alex Van Moerkerckea,b, Priscille Steensmac, Fabian Schweizera,b, Jacob Polliera,b, Ivo Gariboldic, Richard Payned, Robin Vanden Bosschea,b, Karel Miettinena,b, Javiera Espozc, Purin Candra Purnamac, Franziska Kellnerd, Tuulikki Seppänen-Laaksoe, Sarah E. O’Connord, Heiko Rischere, Johan Memelinkc, and Alain Goossensa,b,1

·  aDepartment of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), 9052 Gent, Belgium;

·  bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium;

·  cInstitute of Biology, Leiden University, 2300 RA Leiden, The Netherlands;

·  dDepartment of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom;

·  eVTT Technical Research Centre of Finland Ltd., FIN-02044 VTT, Espoo, Finland

Significance

Terpenoids are the largest group of plant-specialized metabolites and include many valuable bioactive compounds, such as the blockbuster anticancer drugs vincristine and vinblastine, that are monoterpenoid indole alkaloids from the medicinal plant Catharanthus roseus (Madagascar periwinkle). A master regulator was discovered that activates the biosynthesis of the iridoids, the monoterpenoid precursors of vinblastine and vincristine, and the rate-limiting branch in their biosynthetic pathway. This master regulator can be used to boost production of iridoids and monoterpenoid indole alkaloids in C. roseus cell cultures and thus represents an interesting tool for the metabolic engineering of the sustainable production of these high-value compounds in cultures of the endogenous plant species.

Abstract

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix–loop–helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.

 

See: http://www.pnas.org/content/112/26/8130.abstract.html?etoc

PNAS June 30, 2015 vol. 112 no. 26 8130-8135

 

Fig. 1.

BIS1 is coexpressed with the iridoid pathway genes in C. roseus. (A) Pathways leading to the production of MIAs and triterpenoids in C. roseus. Genes activated by BIS1 and ORCA3 overexpression are boxed in blue and green, respectively. 7DLGT, 7-deoxyloganetic acid glucosyl transferase; 7DLH, 7-deoxyloganic acid hydroxylase; 8HGO, 8-hydroxygeraniol oxidoreductase; AAS, α-amyrin synthase; AO, amyrin oxidase; DXR, 1-deoxy-5-xylulose-5-phosphate reductase; FPP, farnesyl pyrophosphate; G3P, glyceraldehyde 3-phosphate; G8O, geraniol-8-oxidase; GES, geraniol synthase; GPP, geranyl diphosphate; HDS, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; IO, iridoid oxidase; IS, iridoid synthase; LAMT, loganic acid O-methyltransferase; MECS, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate synthase; MEP, 2-C-methyl-d-erythritol 4-phosphate; MVA, mevalonate; SLS, secologanin synthase; STR, strictosidine synthase; TDC, tryptophan decarboxylase. (B and C) Coexpression analysis of BIS1, ORCA3, MYC2, and the known MIA and triterpenoid pathway genes. The average linkage hierarchical clustering with Pearson correlation was used. FKPM values along with Caros and gene ID can be found in SI Appendix, Tables S1 and S2. Blue and yellow denote relative down-regulation and up-regulation, respectively. (B) Selected RNA-Seq data from the Medicinal Plant Genomics Resource (medicinalplantgenomics.msu.edu). Values were normalized to the seedling reads. IM, immature leaf; ML, mature leaf. (C) RNA-Seq from the Online Resource for Community Annotation of Eukaryotes (ORCAE) database from the SmartCell consortium (bioinformatics.psb.ugent.be/orcae/overview/Catro). Values were normalized to the control cell culture (CC_c). CC_JA, JA-treated cell culture; CC_O2 and CC_O3, cell culture overexpressing ORCA2 and ORCA3. Genes indicated in gray were not expressed in the cell culture.

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