Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.     

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in h?rhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.     

Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 mol l^–1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.     

Effect of phytohormones on growth and alkaloid accumulation by a Catharanthus roseus cell suspension cultures fed with alkaloid precursors tryptamine and loganin

This work presents a study of the effect of different phytohormones on growth and accumulation of terpenoid indole alkaloids in a Catharanthus roseus cell suspension culture upon feeding with the precursors loganin and tryptamine. The phytohormones tested were 2,4-dichlorophenoxyacetic acid, salicylic acid, methyl jasmonate and abscisic acid. Among these only methyl jasmonate enhanced the accumulation of alkaloids. Abscisic acid did not enhance the accumulation of alkaloids but delayed the catabolism of strictosidine.     

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.     

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.     

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     

Transient effects of overexpressing anthranilate synthase alpha and beta subunits in Catharanthus roseus hairy roots

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, h?rhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.     

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.     

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.     

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.     

Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.     

Precursor limitations in methyl jasmonate-induced Catharanthus roseus cell cultures

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 μM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1–1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.     

Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus

Cell suspension cultures (cell line No 615) of Catharanthus roseus cv. Little Delicata responded to elicitor treatment by accumulating monoterpenoid indole alkaloids and phenolic compounds. The excretion of phenols into the culture medium resulted from the induction of the branch-point enzyme phenylalanine ammonia lyase. The accumulation of alkaloids, however, occurred several hours earlier than the elicitor-mediated induction of tryptophan decarboxylase through which shikimate pathway intermediates are channelled into tryptamine and related indole alkaloids. The results indicate that both pathways for phenol and indole alkaloid biosynthesis responded to elicitor treatment and that no obvious causal relationship between pathways could be deduced from this study.Abbreviations PAL phenylalanine ammonia lyase - TDC tryptophan decarboxylase Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Biotechnology for the production of plant secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.     

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.     

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alkaloid formation in cell suspension cultures of Tabernaemontana divaricata after feeding of tryptamine and loganin

A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors was converted into indole alkaloids such as strictosidine, vallesamine, O-acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of subculturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways.