Production Of Terpenoid Indole Alkaloids Research Articles

Microbial allies: exploring fungal endophytes for biosynthesis of terpenoid indole alkaloids.

Terpenoid indole alkaloids (TIAs) are natural compounds found in medicinal plants that exhibit various therapeutic activities, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, anti-helminthic, and anti-tumor properties. However, the production of these alkaloids in plants is limited, and there is a high demand for them due to the increasing incidence of cancer cases. To address this research gap, researchers have focused on optimizing culture media, eliciting metabolic pathways, overexpressing genes, and searching for potential sources of TIAs in organisms other than plants. The insufficient number of essential genes and enzymes in the biosynthesis pathway is the reason behind the limited production of TIAs. As the field of natural product discovery from biological species continues to grow, endophytes are being investigated more and more as potential sources of bioactive metabolites with a variety of chemical structures. Endophytes are microorganisms (fungi, bacteria, archaea, and actinomycetes), that exert a significant influence on the metabolic pathways of both the host plants and the endophytic cells. Bio-prospection of fungal endophytes has shown the discovery of novel, high-value bioactive compounds of commercial significance. The discovery of therapeutically significant secondary metabolites has been made easier by endophytic entities' abundant but understudied diversity. It has been observed that fungal endophytes have better intermediate processing ability due to cellular compartmentation. This paper focuses on fungal endophytes and their metabolic ability to produce complex TIAs, recent advancements in this area, and addressing the limitations and future perspectives related to TIA production.

Exploration of the Mechanisms of Differential Indole Alkaloid Biosynthesis in Dedifferentiated and Cambial Meristematic Cells of Catharanthus roseus Using Transcriptome Sequencing.

Catharanthus roseus produces terpenoid indole alkaloids (TIAs) of high medicinal importance. The current research focuses on finding an efficient production system such as cell suspension cultures for high TIA concentrations. Catharanthus roseus cambial meristematic cells (CMCs) offer multiple advantages over dedifferentiated cells (DDCs) regarding growth, homogeneity, and shear resistance. Our lab has established a CMC culture system induced by C. roseus cambium. We determined the concentrations of TIAs in CMCs and DDCs. CMCs produced significantly higher concentrations of total alkaloids, vindoline, vinblastine, catharanthine, and ajmalicine as compared to DDCs. We then performed Illumina HiSeq transcriptome sequencing of CMCs and DDCs and explored the differential transcriptomic signatures. Of the 96,004 unigenes, 9,564 were differentially expressed between the 2 cell suspension types. These differentially expressed genes (DEGs) were enriched in 137 KEGG pathways. Most importantly, genes from the indole alkaloid biosynthesis and the upstream pathways i.e., tryptophan metabolism, monoterpenoid biosynthesis, tropane, piperidine, and pyridine alkaloid biosynthesis, and terpenoid backbone biosynthesis showed differential transcriptomic signatures. Remarkably, the expression of genes associated with plant hormone biosynthesis, signaling, and MAPK signaling pathways was relatable to the different TIA concentrations in CMCs and DDCs. These results put forward multiple target genes, transcription factors, and regulators to develop a large-scale TIA production system using C. roseus CMCs.

Repression of ZCT1, ZCT2 and ZCT3 affects expression of terpenoid indole alkaloid biosynthetic and regulatory genes.

Terpenoid indole alkaloids (TIAs) include several valuable pharmaceuticals. As Catharanthus roseus remains the primary source of these TIA pharmaceuticals, several research groups have devoted substantial efforts to increase production of these compounds by C. roseus. Efforts to increase TIA production by overexpressing positive regulators of TIA biosynthetic genes have met with limited success. This limited success might be due to the fact that overexpression of several positive TIA regulators turns on expression of negative regulators of TIA biosynthetic genes. Consequently, a more effective approach for increasing expression of TIA biosynthetic genes might be to decrease expression of negative regulators of TIA biosynthetic genes. Towards this end, an RNAi construct was generated that expresses a hairpin RNA carrying nucleotide fragments from three negative transcriptional regulators of TIA genes, ZCT1, ZCT2 and ZCT3, under the control of a beta-estradiol inducible promoter. Transgenic C. roseus hairy root lines carrying this ZCT RNAi construct exhibit significant reductions in transcript levels of all three ZCT genes. Surprisingly, out of eight TIA biosynthetic genes analyzed, seven (CPR, LAMT, TDC, STR, 16OMT, D4H and DAT) exhibited decreased rather than increased transcript levels in response to reductions in ZCT transcript levels. The lone exception was T19H, which exhibited the expected negative correlation in transcript levels with transcript levels of all three ZCT genes. A possible explanation for the T19H expression pattern being the opposite of the expression patterns of the other TIA biosynthetic genes tested is that T19H shunts metabolites away from vindoline production whereas the products of the other genes tested shunt metabolites towards vindoline metabolism. Consequently, both increased expression of T19H and decreased expression of one or more of the other seven genes tested would be expected to have similar effects on flux through the TIA pathway. As T19H expression is lower in the ZCT RNAi hairy root lines than in the control hairy root line, the ZCTs could act directly to inhibit expression of T19H. In contrast, ZCT regulation of the other seven TIA biosynthetic genes tested is likely to occur indirectly, possibly by the ZCTs turning off expression of a negative transcriptional regulator of some TIA genes. In fact, transcript levels of a negative TIA transcriptional regulator, GBF1, exhibited a strong, and statistically significant, negative correlation with transcript levels of ZCT1, ZCT2 and ZCT3. Together, these findings suggest that the ZCTs repress expression of some TIA biosynthetic genes, but increase expression of other TIA biosynthetic genes, possibly by turning down expression of GBF1.

ZCTs knockdown using antisense LNA GapmeR in specialized photomixotrophic cell suspensions of Catharanthus roseus: Rerouting the flux towards mono and dimeric indole alkaloids.

The online version contains supplementary material available at 10.1007/s12298-021-01017-y.

Effect of abiotic elicitation and pathway precursors feeding over terpenoid indole alkaloids production in multiple shoot and callus cultures of Catharanthus roseus

Catharanthus roseus is a well-known herb with great pharmaceutical value for being the only source of natural antineoplastic drugs vinblastine and vincristine. The terpenoid indole alkaloids (TIAs) biosynthetic pathway responsible for the production of these antineoplastic drug molecules is under a strict spatiotemporal regulation that requires five different cellular as well as at least four intracellular compartmentations for its completion. Therefore, several efforts were made to investigate the cell and tissue cultures of Catharanthus roseus to establish as an alternative source of TIAs production. Cell suspension, hairy roots and callus cultures of Catharanthus roseus do not provide the required cellular complexity for the completion of entire TIAs pathway, therefore, failed to produce vinblastine and vincristine. However, the multiple shoot cultures do provide the required cellular complexity to complete the entire TIAs pathway. Therefore, in the present study, the multiple shoot cultures along with callus cultures were subjected to the abiotic elicitors and TIAs pathway precursors feeding for the first time to chase the effect of these abiotic elicitors and pathway precursors on the production of major TIAs of Catharanthus roseus. The multiple shoot cultures treated with TIAs pathway precursor tryptamine 300 mg/L accumulated highest vinblastine content (0.0277% dry wt) followed by tryptophan feeding at 300 mg/L (0.0180% dry wt) and 500 mg/L (0.0175% dry wt) whereas, callus cultures failed to produce vinblastine.

Effect of Aspergillus flavus Fungal Elicitor on the Production of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells.

This study reported the inducing effect of Aspergillus flavus fungal elicitor on biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus cambial meristematic cells (CMCs) and its inducing mechanism. According to the results determined by HPLC and HPLC-MS/MS, the optimal condition of the A. flavus elicitor was as follows: after suspension culture of C. roseus CMCs for 6 day, 25 mg/L A. flavus mycelium elicitor were added, and the CMC suspensions were further cultured for another 48 h. In this condition, the contents of vindoline, catharanthine, and ajmaline were 1.45-, 3.29-, and 2.14-times as high as those of the control group, respectively. Transcriptome analysis showed that D4H, G10H, GES, IRS, LAMT, SGD, STR, TDC, and ORCA3 were involved in the regulation of this induction process. The results of qRT-PCR indicated that the increasing accumulations of vindoline, catharanthine, and ajmaline in C. roseus CMCs were correlated with the increasing expression of the above genes. Therefore, A. flavus fungal elicitor could enhance the TIA production of C. roseus CMCs, which might be used as an alternative biotechnological resource for obtaining bioactive alkaloids.

Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis.

Biosynthesis of medicinally valuable terpenoid indole alkaloids (TIAs) in Catharanthus roseus is regulated by transcriptional activators such as the basic helix-loop-helix factor CrMYC2. However, the transactivation effects are often buffered by repressors, such as the bZIP factors CrGBF1 and CrGBF2, possibly to fine-tune the accumulation of cytotoxic TIAs. Questions remain as to whether and how these factors interact to modulate TIA production. We demonstrated that overexpression of CrMYC2 induces CrGBF expression and results in reduced alkaloid accumulation in C. roseus hairy roots. We found that CrGBF1 and CrGBF2 form homo- and heterodimers to repress the transcriptional activities of key TIA pathway gene promoters. We showed that CrGBFs dimerize with CrMYC2, and CrGBF1 binds to the same cis-elements (T/G-box) as CrMYC2 in the target gene promoters. Our findings suggest that CrGBFs antagonize CrMYC2 transactivation possibly by competitive binding to the T/G-box in the target promoters and/or protein-protein interaction that forms a non-DNA binding complex that prevents CrMYC2 from binding to its target promoters. Homo- and heterodimer formation allows fine-tuning of the amplitude of TIA gene expression. Our findings reveal a previously undescribed regulatory mechanism that governs the TIA pathway genes to balance metabolic flux for TIA production in C. roseus.

ПОЛУЧЕНИЕ ЛЕКАРСТВЕННОГО РАСТИТЕЛЬНОГО СЫРЬЯ КАТАРАНТУСА РОЗОВОГО С ИСПОЛЬЗОВАНИЕМ СВЕТОДИОДНОГО ОСВЕЩЕНИЯ В УСЛОВИЯХ ЗАКРЫТОГО ГРУНТА

Сatharanthus roseus G. Don is an important medicinal plant which accumulates pharmacologically important terpenoid indole alkaloids (TIA) and phenolic compounds. TIA of C. roseus includes more than 130 substances. Ajmalicine and serpentine are used for arterial hypertension treatment. Vinblastine and vincristine are well known as antineoplastic drugs used in chemotherapy of cancer diseases.Light-Emitting Diodes (LEDs) are modern sources of energy allowing production of the light wavelengths corresponding to the absorption peaks of plants photosynthesis and photomorphogenesis.Influence of different LED light regimes on the growth and biosynthesis processes in C. roseus was studied. Our results have demonstrated that some LEDs light regimes strongly stimulated C. roseus growth, flowering and biomass production. fullerene-arginine were shown to regulate seeds germination, seedlings growth, and water transport. We found different effects of LEDs light regimes fullerene derivatives on TIA and phenolic compounds accumulations in plant tissues. The LEDs light regimes stimulating of TIA production were determinated. It was observed that phenolic and antiradical scavenging compounds content in barley seedlings could be reduced in presence of fullerenol or fullerene-arginine. Physiological and biochemical effects of fullerene derivatives were depended on their concentration as well as temperature and light conditions of plant growth.The results of this work can be used in the creation of techniques for optimization of ornamental and medicinal plants cultivation.

Biotechnology of the medicinal plant Rhazya stricta: a little investigated member of the Apocynaceae family.

Rhazya stricta Decne. (Apocynaceae) is an important medicinal plant that is widely distributed in the Middle East and Indian sub-continent. It produces a large number of terpenoid indole alkaloids (TIAs) some of which possess important pharmacological properties. However, the yields of these compounds are very low. Establishment of a reliable, reproducible and efficient transformation method and induction of hairy roots system is a vital prerequisite for application of biotechnology in order to improve secondary metabolite yields. In the present review, recent biotechnological attempts and advances in TIAs production through transformed hairy root cultures in R. stricta are reviewed to draw the attention to its metabolic engineering potential.

Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

Artemisinic Acid Serves as a Novel ORCA3 Inducer to Enhance Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells

To investigate the effect of artemisinic acid (AA) on improving the production of terpenoid indole alkaloids (TIAs) of Catharanthus roseus cambial meristematic cells (CMCs), feeding AA to C. roseus CMCs caused 2.35-fold and 2.51-fold increases in the production of vindoline and catharanthine, respectively, compared with those of the untreated CMCs. qRT-PCR experiments showed that AA resulted in a 1.36-8.52 fold increase in the transcript levels of several related genes, including octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3), tryptophan decarboxylase (TDC), strictosidine synthase (STR) and desacetoxyvindoline 4-hydroxylase (D4H). However, no effect was observed on the concentration of either jasmonic acid (JA), or the octadecanoid-pathway inhibitors block TIA accumulation caused by AA. The results indicated that AA might serve as a novel ORCA3 inducer to manipulate biosynthesis of TIAs in C. roseus CMCs via an unknown mechanism.

Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0794-4) contains supplementary material, which is available to authorized users.

Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production.

Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots. Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83% for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.

Effects of β-cyclodextrin and methyl jasmonate on the production of vindoline, catharanthine, and ajmalicine in Catharanthus roseus cambial meristematic cell cultures.

Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

Metabolic alterations and distribution of five-carbon precursors in jasmonic acid-elicited Catharanthus roseus cell suspension cultures

In Catharanthus roseus cell cultures, the terpenoid moiety is considered as the limiting factor in terpenoid indole alkaloid (TIA) production. The pathway of terpenoidal precursors in TIA is strongly linked with other terpenoid pathways, suggesting that TIA production might be limited by competition for the five-carbon (C5) precursors. This raises the question whether the stimulation of TIA production by certain signal molecules is due to a redistribution of C5 precursors between the associated terpenoid pathways and/or to a total increase of the precursor availability. To investigate the effect of a TIA-increasing signal molecule on C5 distribution, the cell suspension cultures of C. roseus were elicited with jasmonic acid (JA) and the metabolic changes of different terpenoid pathways were evaluated targeting on TIA (monoterpenoid; C10), carotenoids (tetraterpenoid; C40), and phytosterols (triterpenoid; C30). The chromatographic analyses showed that TIA and carotenoid levels almost doubled upon JA elicitation, while phytosterol levels remained constant if compared to the controls. Apparently, both TIA and carotenoid routes benefit from an increased C5 flow in the methyl-erythritol phosphate pathway, and potential export of C5 precursors to the cytosolic terpenoid routes, e.g., towards the phytosterols is minimal. However, the relative composition of individual compounds within each group remains similar when comparing elicited and control cells. This suggests that the increased production of TIA and carotenoid upon JA elicitation is predominantly caused by an increase of the precursor availability rather than due to a redistribution of C5 precursors between the associated terpenoid pathways. Furthermore, NMR-based metabolomics analysis showed a discrimination of JA-elicited and control cells between 24 and 72 h after treatments with significant changes in levels of strictosidine, malic acid, and sucrose. This study portrays metabolic alterations upon JA elicitation and channeling of C5 precursors in different terpenoid biosynthetic pathways, which provides a knowledge platform for developing strategies to engineer fluxes in a complex biosynthetic network in order to obtain high TIA-producing C. roseus cell lines.

Indole alkaloids from Catharanthus roseus: bioproduction and their effect on human health.

Catharanthus roseus is a medicinal plant belonging to the family Apocynaceae which produces terpenoid indole alkaloids (TIAs) of high medicinal importance. Indeed, a number of activities like antidiabetic, bactericide and antihypertensive are linked to C. roseus. Nevertheless, the high added value of this plant is based on its enormous pharmaceutical interest, producing more than 130 TIAs, some of which exhibit strong pharmacological activities. The most striking biological activity investigated has been the antitumour effect of dimeric alkaloids such as anhydrovinblastine, vinblastine and vincristine which are already in pre-, clinical or in use. The great pharmacological importance of these indole alkaloids, contrasts with the small amounts of them found in this plant, making their extraction a very expensive process. To overcome this problem, researches have looked for alternative sources and strategies to produce them in higher amounts. In this sense, intensive research on the biosynthesis of TIAs and the regulation of their pathways has been developed with the aim to increase by biotechnological approaches, the production of these high added value compounds. This review is focused on the different strategies which improve TIA production, and in the analysis of the beneficial effects that these compounds exert on human health.

Ornamental exterior versus therapeutic interior of Madagascar periwinkle (Catharanthus roseus): the two faces of a versatile herb.

Catharanthus roseus (L.) known as Madagascar periwinkle (MP) is a legendary medicinal plant mostly because of possessing two invaluable antitumor terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The plant has also high aesthetic value as an evergreen ornamental that yields prolific blooms of splendid colors. The plant possesses yet another unique characteristic as an amiable experimental host for the maintenance of the smallest bacteria found on earth, the phytoplasmas and spiroplasmas, and serves as a model for their study. Botanical information with respect to synonyms, vernacular names, cultivars, floral morphology, and reproduction adds to understanding of the plant while the geography and ecology of periwinkle illustrate the organism's ubiquity. Good agronomic practices ensure generous propagation of healthy plants that serve as a source of bioactive compounds and multitudinous horticultural applications. The correlation between genetic diversity, variants, and TIA production exists. MP is afflicted with a whole range of diseases that have to be properly managed. The ethnobotanical significance of MP is exemplified by its international usage as a traditional remedy for abundant ailments and not only for cancer. TIAs are present only in micro quantities in the plant and are highly poisonous per se rendering a challenge for researchers to increase yield and reduce toxicity.

Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids.

Hairy root cultures of Rauwolfia serpentina induced by Agrobacterium rhizogenes have been investigated extensively for the production of terpenoid indole alkaloids. Various biotechnological developments, such as scaling up in bioreactors, pathway engineering etc., have been explored to improve their metabolite production potential. These hairy roots are competent for regenerating into complete plants and show survival and unaltered biosynthetic potential during storage at low temperature. This review provides a comprehensive account of the hairy root cultures of R. serpentina, their biosynthetic potential and various biotechnological methods used to explore the production of pharmaceutically important terpenoid indole alkaloids. The review also indicates how biotechnological endeavors might improve the future progress of research for production of alkaloids using Rauwolfia hairy roots.

Metabolic engineering and elicitation of pharmaceutically active metabolites in Rhazya stricta (Apocynaceae)

Rhazya stricta Decne., a small evergreen shrub native to the Middle East and Indian sub-continent, is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). The plant has been in the focus of phytochemical, pharmacological and ethnobotanical studies due to its long use in folk medicine to treat a variety of ailments. More recently especially the antimicrobial and anticancer properties of certain constituents (Fig. 1) have been established. TIAs are formed by the condensation of the indole and iridoid moieties catalysed by strictosidine synthase (STR) to form the precursor strictosidine. High STR activity associated with the induction of rate-limiting enzyme activities of the iridoid pathway (GES, G10 H) favours TIA production. Biosynthetic steps in the TIA pathway are co-ordinately regulated at the level of transcription of the structural genes by transcription factors (e.g. orca3).

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.