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.     

Enzymatic formation of intermediates in the biosynthests of ajmalicine: Strictosidine and cathenamine

Pre-purified enzymes isolated from Catharanthus roseus suspension cultures synthesize strictosidine and cathenamine from tryptamine and secologanin. Whereas strictosidine showed metabolic activity, cathenamine accumulates during the cell-free incubations in the absence of reduced pyridine nucleotides. In the presence of δ-d-gluconolactone (0.1 M), strictosidine accumulates in a yield of ca 50%. Optimum conditions for its accumulation in crude extracts were found to be at pH 4.1, 0.25 mM tryptamine and 1.25 mM secologinin. Strictosidine synthase is stable for more than 1.5 months at 4°. The optimum conditions for the enzymatic synthesis of cathenamine are 1.54 mM tryptamine and 7.7 mM secologanin at pH 7.5. In the presence of NH₄⁺ the formation of the latter alkaloid decreases due to the synthesis of unidentified compounds.     

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     

Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.     

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.     

Effects of alkaloid precursor feeding on a Camptotheca acuminata cell line

To better understand the biosynthesis of Camptotheca acuminata alkaloids, the effect on camptothecin production of feeding with potential precursors of biosynthesis was studied (i.e., tryptamine and loganin combined, secologanin, and strictosidine). Two key enzymes in alkaloid biosynthesis 〚i.e., tryptophan decarboxylase (TDC; EC 4.1.1.28) and strictosidine synthase (STR; EC 4.3.3.2)〛 were also studied. The analyses were conducted using a C. acuminata CG1 cell line that does not produce alkaloids, which could be useful in better understanding the biosynthetic pathway and in identifying possible limiting factors. The activity of TDC was 5 pkat mg–1; the activity of STR was 1.1 pkat mg^–1. Feeding with strictosidine revealed that this precursor is easily biotransformed by two enzymes (i.e., a hydroxylase and a dehydrogenase) in hydroxystrictosidine and didehydrostrictosidine, but camptothecin was never detected. The indole pathway and the low level of STR activity could be limiting factors in the production of camptothecin in the cell line used.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of strictosidine using<Emphasis Type="Italic">lonicera japonica</Emphasis> leaf extracts and recombinant yeast

Strictosidine is a key intermediate in the biosynthesis of the terpenoid indole alkaloid (T1A) pathway. It results from a condensation reaction, catalyzed by strictosidine synthase (STR), between tryptamine and secologanin. We have now developed a useful method, based on enzyme-assisted synthesis, to produce strictosidine. Our procedure utilizes leaf extracts from Japanese honeysuckleLonicera japonica Thunb. as a secologanin source. In these experiments, an enzyme extract was prepared from transgenic yeastSaccharomyces cerevisiae that expresses theCatharanthus roseus STR (CrSTR) coding region. Strictosidine was then isolated with a 38% yield based on the initial amount of tryptamine in the enzymatic reaction.     

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.     

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.     

Assay of strictosidine synthase from plant cell cultures by high-performance liquid chromatography

An HPLC assay is described for the enzyme strictosidine synthase in which the formation of strictosidine and the decrease of tryptamine can be followed at the same time. In cell cultures of Catharanthus roseus significant amounts of strictosidine glucosidase activity were detected. In crude preparations, the strictosidine synthase reaction is therefore best measured by the secologanin-dependent decrease of tryptamine. In this way, the specific synthase activity in a cell free extract was found to be 56 pkat/mg of protein. Inclusion of 100 mM D(+)-gluconic acid-delta-lactone in the incubation mixture inhibited 75% of the glucosidase activity, without inhibiting the synthase activity. The synthase activity was readily separated from the glucosidase activity by gel filtration on Sephadex G-75 or Ultrogel AcA-44. Cell cultures of Tabernaemontana orientalis did not contain measurable amounts of strictosidine glucosidine activity. The specific strictosidine synthase activity was 130-200 pkat/mg of protein during the growth of this cell culture. Strictosidine synthase is stable at -20 degrees C for at least 2 months.     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

Why are the terpenoid indole alkaloids of type I homochiral?

In the presence of the enzyme strictosidine synthase, the coupling reaction of secologanin and tryptamine is completely stereoselective and affords strictosidine with 3S configuration, exclusively. The stereoselectivity is transferred and retained in most indole alkaloids of type I in which C-3 is not involved in subsequent reactions. By using results of model reactions, the stereoselectivity was interpreted by the bulkiness of the enzyme temporarily attached to the N-4 atom in the formation of the indolenine intermediate. 3S configuration is kept in the subsequent 1,2-rearrangement into the beta-carboline structure. In the formation of the oxindole derivatives, the 3S configuration is preferred, but not necessarily complete.     

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells. Received: 25 February 1996 / Revision received: 16 August 1996 / Accepted: 30 September 1996     

3D-Structure and function of strictosidine synthase--the key enzyme of monoterpenoid indole alkaloid biosynthesis

Strictosidine synthase (STR; EC 4.3.3.2) plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicinal plant Rauvolfia serpentina represents the first example of a six-bladed four-stranded beta-propeller fold from the plant kingdom. Moreover, the architecture of the enzyme-substrate and enzyme-product complexes reveals deep insight into the active centre and mechanism of the synthase highlighting the importance of Glu309 as the catalytic residue. The present review describes the 3D-structure and function of R. serpentina strictosidine synthase and provides a summary of the strictosidine synthase substrate specificity studies carried out in different organisms to date. Based on the enzyme-product complex, this paper goes on to describe a rational, structure-based redesign of the enzyme, which offers the opportunity to produce novel strictosidine derivatives which can be used to generate alkaloid libraries of the N-analogues heteroyohimbine type. Finally, alignment studies of functionally expressed strictosidine synthases are presented and the evolutionary aspects of sequence- and structure-related beta-propeller folds are discussed.