A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

[Advance in biosynthesis of terpenoid indole alkaloids and its regulation in Catharanthus roseus].

Catharanthus roseus can produce a variety of terpenoid indole alkaloids (TIA), most of which exhibit strong pharmacological activities. Hence, biosynthesis and regulation of TIA have received recent attention. 3α (S)-strictosidine is an important node in TIA biosynthesis, which is a condensation product of secologanin and tryptamine. The former is produced in iridoid pathway, and the latter is produced in indole pathway. Vindoline and catharanthine, which are produced respectively by 3α (S)-strictosidine via multi-step enzymatic reaction, can form α-3, 4-anhydrovinblastine by the condensation reaction. Then, vinblastine and vincristine are generated from α-3, 4-anhydrovinblastine. Many transcription factors are involved in the regulation of TIA synthesis, such as AP2/ERF and WRKY. Illumination of biosynthetic pathway has laid a foundation for the study of synthetic biology. Today, 3α (S)-strictosidine and vindoline have been synthesized in heterologous hosts Saccharomyces cerevisiae.Research about synthetic biology and the regulation mechanisms will provide a guidance for the production and development of TIA drugs in C. roseus.

Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Biosynthesis of the therapeutically valuable terpenoid indole alkaloids (TIAs), in the medicinal plant Catharanthus roseus, is one of the most elaborate and complex metabolic processes. Although genomic and transcriptomic resources have significantly accelerated gene discovery in the TIA pathway, relatively few genes of transcription factors (TFs) have been identified and characterized thus far. Systematic identification of TFs and elucidation of their functions are crucial for understanding TIA pathway regulation. The successful discovery of TFs in the TIA pathway has relied mostly on three different approaches, (1) identification of cis-regulatory motifs (CRMs) present in the pathway gene promoters as they often provide clues on potential TFs that bind to the promoters, (2) co-expression analysis, based on the assumption that TFs regulating a metabolic or developmental pathway exhibit similar spatiotemporal expression as the pathway genes, and (3) isolation of homologs of TFs known to regulate structurally similar or diverse specialized metabolites in different plant species. TFs regulating TIA pathway have been isolated using either an individual or a combination of the three approaches. Here we describe transcriptome-based coexpression analysis and cis-element determination to identify TFs in C. roseus. In addition, we describe the protocols for generation of transgenic hairy roots, Agrobacterium infiltration of flowers, and electrophoretic mobility shift assay (EMSA). The methods described here are useful for the identification and characterization of potential TFs involved in the regulation of special metabolism in other medicinal plants.

Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus.

Terpenoid indole alkaloid (TIA) biosynthetic pathway of Catharanthus roseus possesses the major attention in current metabolic engineering efforts being the sole source of highly expensive antineoplastic molecules vinblastine and vincristine. The entire TIA pathway is fairly known at biochemical and genetic levels except the pathway steps leading to biosynthesis of catharanthine and tabersonine. To increase the in-planta yield of these antineoplastic metabolites for the pharmaceutical and drug industry, extensive plant tissue culture-based studies were performed to provide alternative production systems. However, the strict spatiotemporal developmental regulation of TIA biosynthesis has restricted the utility of these cultures for large-scale production. Therefore, the present study was performed to enhance the metabolic flux of TIA pathway towards the biosynthesis of vinblastine by overexpressing two upstream TIA pathway genes, tryptophan decarboxylase (CrTDC) and strictosidine synthase (CrSTR), at whole plant levels in C. roseus. Whole plant transgenic of C. roseus was developed using Agrobacterium tumefaciens LBA1119 strain having CrTDC and CrSTR gene cassette. Developed transgenic lines demonstrated up to twofold enhanced total alkaloid production with maximum ninefold increase in vindoline and catharanthine, and fivefold increased vinblastine production. These lines recorded a maximum of 38-fold and 65-fold enhanced transcript levels of CrTDC and CrSTR genes, respectively.

Genetic engineering approach using early Vinca alkaloid biosynthesis genes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus.

Catharanthus roseus today occupies the central position in ongoing metabolic engineering efforts in medicinal plants. The entire multi-step biogenetic pathway of its very expensive anticancerous alkaloids vinblastine and vincristine is fairly very well dissected at biochemical and gene levels except the pathway steps leading to biosynthesis of monomeric alkaloid catharanthine and tabersonine. In order to enhance the plant-based productivity of these pharma molecules for the drug industry, cell and tissue cultures of C. roseus are being increasingly tested to provide their alternate production platforms. However, a rigid developmental regulation and involvement of different cell, tissues, and organelles in the synthesis of these alkaloids have restricted the utility of these cultures. Therefore, the present study was carried out with pushing the terpenoid indole alkaloid pathway metabolic flux towards dimeric alkaloids vinblastine and vincristine production by over-expressing the two upstream pathway genes tryptophan decarboxylase and strictosidine synthase at two different levels of cellular organization viz. callus and leaf tissues. The transformation experiments were carried out using Agrobacterium tumefaciens LBA1119 strain having tryptophan decarboxylase and strictosidine synthase gene cassette. The callus transformation reported a maximum of 0.027% dry wt vindoline and 0.053% dry wt catharanthine production, whereas, the transiently transformed leaves reported a maximum of 0.30% dry wt vindoline, 0.10% catharanthine, and 0.0027% dry wt vinblastine content.