The indole alkaloid tryptamine produced in transgenic Petunia hybrida

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine, an anti-insect compound. This TDC cDNA was transformed and expressed in transgenic Petunia hybrida under the control of the strong and constitutive 35S promoter from cauliflower mosaic virus. Kanamycin screening and Southern hybridization with the TDC cDNA confirmed plant transformation. Northern analysis indicated greater TDC mRNA accumulation in transgenic plants compared to non-transformed plants. Additionally, eight-fold more tryptamine accumulated in leaves of kanamycin resistant transgenic plants compared to non-transformed plants. Flower petals from the transgenic plants contained lower tryptamine levels than their leaves. Because tryptamine titers were higher in transformed plants compared to controls, over-expression of the TDC enzyme may partially overcome endogenous tryptamine catabolism and/or other negative biosynthetic regulation. Future alteration of tryptamine breakdown in Petunia may further increase total endogenous tryptamine concentrations, potentially discouraging insect reproduction on these transgenic plants.     

Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. Petit Havana SR1 F₁-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.     

Tryptophan Decarboxylase,Tryptamine, and Reproduction of the Whitefly

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.     

Molecular cloning and expression of tryptophan decarboxylase from Mitragyna speciosa

Tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine in mitragynine biosynthesis via the shikimate pathway. Using the rapid amplification of cDNA ends (RACE) technique, the gene encoding TDC from Mitragyna speciosa was cloned (designated as MsTDC). The MsTDC cDNA contained an open reading frame (ORF) of 1,521 base pairs (bp) encoding 506 amino acid residues. It had a pyridoxal-phosphate (PLP)-binding site at the amino acid position 313–334 residues. The MsTDC showed homology of 68–76 % to the TDC of other plants. Heterologous expression in Escherichia coli afforded the soluble proteins as an apparent band of 57 kDa as judged by SDS-PAGE. Expression of the MsTDC in M. speciosa hairy roots under the 35S promoter was performed by insertion of MsTDC into pCAMBIA1300-gfp. The transgenic hairy root lines were detected by fluorescence microscopy and showed an increased accumulation of tryptamine.     

Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K_m relation to the substrate tryptophan (690 µM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K_m values with corresponding substrates. This suggests that the biosynthesis of serotonin or serotonin-derived secondary metabolites is restricted to cellular stages when high tryptophan levels are present.Key words: feruloylserotonin, serotonin, tryptamine, tryptamine 5-hydroxylase, tryptophan, tryptophan biosynthesis, tryptophan decarboxylaseSerotonin is found in a broad range of plants and is abundant in reproductive organs, such as fruits and seeds.^1–3 Even though many physiological roles for serotonin in plants have been proposed,^2–7 its actual roles have yet to be examined in detail using molecular, biochemical and genetic approaches. In plants, serotonin is synthesized by two enzymes: tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). TDC decarboxylates tryptophan into tryptamine, after which T5H hydroxylates tryptamine into serotonin.^8–10 TDC expresses at an undetectable level in rice leaves, whereas T5H expresses constitutively.^11,12     

Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) upon pathogen infection

It has been suggested that serotonin biosynthesis is regulated by tryptophan decarboxylase (TDC) in plants. To determine if TDC plays a rate-limiting role in serotonin biosynthesis, two TDC genes, PepTDC1 and PepTDC2, were cloned from pepper (Capsicum annuum L.) fruits infected with anthracnose fungus and their expression was then examined in various organs, including fruit that had been treated with the fungus or various chemicals. PepTDC1 expression was highly induced in pepper fruits after treatment with fungus and ethylene, while PepTDC2 was constitutively expressed at low levels in all pepper tissues. Additionally, predominant induction of PepTDC1 mRNA and TDC enzyme activity was detected in the unripe-green fruit, but not in the ripe-red fruit upon pathogen infection. Higher expression of TDC in unripe-green fruit was closely associated with increased levels of tryptamine, serotonin, and serotonin derivatives. However, unlike the enhanced serotonin synthesis, tryptophan levels responded unchanged when challenged with the pathogen in both the unripe-green fruit and the ripe-red fruit. Expression of two key tryptophan biosynthetic genes, anthranilate synthase (ASα) and tryptophan synthase (TSβ), remained unchanged in response to treatment. Also, anthranilate synthase enzyme activity remained steady regardless of pathogen infection. Taken together, these results suggest that the synthesis of serotonin was regulated by the induction of TDC without a simultaneous increase in tryptophan levels in pepper fruits.     

Probing the role of tryptophan-derived secondary metabolism in defense responses against Bipolaris oryzae infection in rice leaves by a suicide substrate of tryptophan decarboxylase

Tryptophan-derived secondary metabolites, including serotonin and its hydroxycinnamic acid amides, markedly accumulate in rice leaves in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by being deposited in cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. In this study, (S)-α-(fluoromethyl)tryptophan (S-αFMT) was utilized to investigate the effects of the inhibition of TDC on the defense responses of rice leaves. S-αFMT, enantiospecifically synthesized from l-tryptophan, effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The inhibition rate increased dependently on the incubation time, indicating that S-αFMT served as a suicide substrate. Treatment of rice seedlings with S-αFMT suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner in B. oryzae-inoculated leaves. The lesions formed on seedlings treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with leaves without S-αFMT treatment. Administrating tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material. In addition, tryptamine administration reduced damage caused by fungal infection. Accordingly, the accumulation of tryptophan-derived secondary metabolites was suggested to be part of the effective defense mechanism of rice.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.     

Overexpression of a tryptophan decarboxylase cDNA inCatharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids inC. roseus by converting tryptophan into tryptamine. Hardly anytdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA ofAgrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which atdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced inC. roseus cells by infecting seedlings with an oncogenicA. tumefaciens strain. In the resulting crown gall tumour calluses harbouring thetdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing thetdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in thetdc cDNA overexpressing crown gall calluses are discussed.     

<Emphasis Type="SmallCaps">l</Emphasis>-tryptophan decarboxylase activity and tryptamine accumulation in callus cultures of <Emphasis Type="Italic">Vinca minor</Emphasis> L.

l-tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyses the formation of tryptamine from tryptophan, and therefore it plays a role in terpenoid indole alkaloids biosynthesis. In this study, TDC activity and tryptamine accumulation were monitored in callus cultures of important medicinal plant Vinca minor L. Callus cultures, established from leaf tissues, were incubated on Murashige and Skoog (MS) medium supplemented with 4.4 μM kinetin and different concentrations (0.44, 1.1, 2.2, 4.4 and 6.6 μM) of naphthaleneacetic acid (NAA), and grown either in the dark or under 16 h photoperiod. When the basal enzyme activity of TDC was determined in these cultures, it was 0.5–0.7 nmol tryptamine mg⁻¹ prot. min⁻¹. Moreover, this activity remained linear over time and over protein concentrations, and with optimum pH levels between 6.5 and 7.5, and an optimum temperature of 35°C. The Michaelis–Menten constant (K_m) for l-tryptophan was 1.3 mM. TDC cofactor, pyridoxal-5′-phosphate (1 mM), increased the enzyme activity. During later stages of callus culture growth cycle, an increase in TDC activity was observed, and this activity depended on culture conditions and age of callus cultures. In addition, TDC activity and tryptamine accumulation in callus cultures were strongly enhanced by light treatment.     

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K _m of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K _m of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g^?1 DW) than the EtOH control (0.183 mg g^?1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g^?1 DW) followed by in bark (0.161 mg g^?1 DW) and young leaf (0.130 mg g^?1 DW), and least in root (0.014 mg g^?1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.     

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans

The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two- to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due to the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestans, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.     

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of -carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

<Emphasis Type="Italic">Withania coagulans</Emphasis> tryptophan decarboxylase gene cloning,heterologous expression,and catalytic characteristics of the recombinant enzyme

Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5′-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher K_m value for tryptophan than for pyridoxal 5′-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.     

Influence of phosphate on the formation of the indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus I. Comparison of enzyme activities and product accumulation

In cell suspension cultures of Catharanthus roseus a rapid accumulation of secondary compounds (tryptamine, indole alkaloids, phenolics) was observed after transfer of the cells into special ‘induction’-media devoid of phosphate and other essential growth factors [11, 14]. The increase of product levels was suppressed in the presence of phosphate which was almost completely taken up from the medium and accumulated by the cells within 48 h after inoculation. The activities of tryptophan decarboxylase (TDC), the first enzyme in indole alkaloid biosynthesis, and of phenyl-alanine ammonia-lyase (PAL), the key enzyme of phenylpropanoid biosynthesis, were influenced differently by phosphate. Whereas the accumulation of phenolics and PAL activity were similarly inhibited by low concentration of phosphate, the medium-induced enhanced activity of TDC was not affected although the product pools were considerably reduced. Some consequences for the regulation of secondary metabolism will be discussed.