Info

Tryptamine

Km (mM) 0.9 immobilized STR (56); 2.3 soluble STR (57); 0.83 soluble STR (58); 8.9d soluble STR (59)

K™, (pkat) 18.9 immobilized STR (56); 130 soluble STR (57); 234" soluble STR (59)

Secologanin

Km (mM) 2.1 immobilized STR (56); 3.4 soluble STR (57)

Vm„ (pkat/mg) 9.1 immobilized STR (56); 55 soluble STR (57)

Stability (half-life time) For crude protein extract (0-70% ammonium sulfate precipitation) 5 hours (60) For immobilized STR at 37°C 68 days (60) For partly purified soluble STR at 37°C 5 hours (60) For partly purified soluble STR at -20°C >2 months (61) For purified STR at -20°C up to 1 year (61)

Substrate specificity Indoles'

Monoterpenoids*

Indoles'

Monoterpenoids' Inhibitors

Tryptamine (100%), 7-methyltryptamine (15%), 6-hydroxytryptamine (9%), 7-fiuorotryptamine (8%), 5-fluorotryptamine (8%),

5-hydroxytryptamine (4%) (57) Secologanin (Km = 3.4 x 10"3 M), 2'-0-methylsecologanin, 3'-0-methylsecologanin (Km = 8 x 10 3 M (57) and Km = 3.6 mM, Vm„ = 14.3) (56), dihydrosecologanin (Km = 3.5 x 10"3) (57)

5-Methyitryptamine, 5-metoxytrptamine, 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, W-methyitryptamine, DL-a-methyltryptamine, a-ethyltryptamine, L-tryptophan, D-tryptophan, phenylethylamine, tyramine, dopamine, homovertrylamine (57) Secologanic acid, iridotrial, tarennoside (57)

p-Chloromercuribenzoate at 1 mM (73% inhibition), no inhibition Ca2+, Co2*, Cu2*, Fe2+, Mn2+, and Zn2+ (57) Weak product inhibition, no substrate inhibition (59) No inhibition by ajmalicine, vindoline, and catharanthine (58)

"STR is demonstrated to be located in vacuole; for discussion see Ref. 48.

"Identity as speculated by De Waal et al. (59) for isomers 4 of 7 (I—IV) of Pfitzner and Zenk (56) and the purified isoenzymes. 'Values are expressed in mM or pkat.

"Average values from six isomers isolated by De Waal et al. (59). "Accepted as substrate.

1 Not accepted as substrate.

Table 3 Studies with (Over)expression of the Tryptophan Decarboxylase (Tdc) Gene

Tdc (51) Origin of the clone Promoter Expressed in Results Tdc (62) Origin of the clone Method Promoter Expressed in Obtained as Results

Tdc (63) Origin of the clone Method Promoter Expressed in Obtained as Results

Tdc (46) Origin of the clone Method Promoter Expressed in Obtained as Results

Catharanthus roseus (L.) G. Don (Apocynaceae) lac

Escherichia coli

No attempt was made to quantify the level of the TDC activity in E. coli.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 51) Agrobacterium-mediated transformation using A. tumefaciens Cauliflower mosaic virus (CaMV) 35S Nicotiana tabacum 'Xanthi' (Solanaceae) Transgenic plants

Leaves of transformed plants had 4 to 45 times more TDC activity than the controls.

Increase in tryptamine accumulation: >1 mg g 1 FW plant with the highest TDC activity, a 260-fold increase over control The growth and development of the transformed plants appeared normal.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 51) Agrobacterium-mediated transformation using A. tumefaciens CaMV 35S

N. tabacum 'Xanthi' (Solanaceae) Transgenic plants

TDC activity in the transformed tobacco was 45 times greater than in the controls and resulted in a >200-fold increase in the free tryptamine and >30-fold in free tyramine pools.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 64) Agrobacterium-med'iated transformation (65) CaMV 35S

Peganum harmala L. (Zygophyllaceae) Cell suspension and hairy root cultures

Despite Increased TDC activity, tryptamine level did not increase due to its conversion Into serotonin. Consequently, serotonin level increased, but no increase In desired harman-type alkaloids was observed.

Tdc (66) Origin of the clone Method Promoter Expressed in Obtained as Results

Tdc (38) Origin of the clone Method Promoter Expressed in Obtained as Results

Tdc (67) Origin of the clone Method Promoter Expressed in Observed as Results

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 67) Agrobacterium-medialedi transformation using A. tumefaciens CaMV 35S

N. tabacum 'Petit Havana' SR1 (Solanaceae) Plants

The plants with highest TDC activity contained 19 pkat/mg protein. Specific activity of AS and CM was not affected by expression of TDC.

Despite increased tryptamlne accumulation, the phenotype of the transgenic plants was normal.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 51) Agrobacterium-mediated transformation using A. tumefaciens CaMV 35S

Brassica napus 'Westar' (Brassicaceae) Transgenic plants

TDC activity in transgenic plants varied and was 4 to 45 times greater than in contrtols

Transgenic plants accumulated tryptamine while correspondingly lower levels of tryptophan-derived indole glucoslnolates were produced in all plant parts compared with controls. Indole glucosinolate content of mature seeds from transgenic plants was only 3% of that found in nontransformed seeds.

Agrobacterium-medialedi transformation using A. tumefaciens CaMV 35S

C. roseus 'Morning Mist' (Apocynaceae) Crown-gall calll

An increase in TDC protein level, TDC activity, and tryptamine content but no significant increase in TIA production were observed.

In 7dc-antisense-containing calli decreased TDC activity was measured.

Table 3 Continued

Tdc (68) Origin of the clone Method Promoter Expressed in Observed as Results

Tdc (69) Origin of the clone Method Promoter Expressed in Observed as

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 51) Agrobacterium-medialed transformation using A. tumefaciens CaMV 35S (as in Ref. 38) Solarium tuberosum 'Désirée' (Solanaceae) Transgenic plants

Phenylpropanoid pathway can be down-regulated by the control of substrate supply created by the introduction of a single gene outside the pathway.

The redirection of tryptophan to tryptamine resulted in a decrease In the levels of tryptophan, phenylalanine, and phenyl-

alanine-derived phenolic compounds in transgenic tubers. Chlorogenic acid was 2- to 3-fold lower in transgenic tubers.

Transgenic tuber became more susceptible to infection with Phytophthora unfastens due to alterations in the substrate levels in the shikimate pathway and phenylpropanoid pathways.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 51)

Agrobacterium-mediated transformation using A. tumefaciens (as in Ref. 62) CAMV 35S

N. tabacum SR1 (Solanaceae) Transgenic plants of the desired harman-type alkaloids did not occur due to the conversion of tryptamine to serotonin (Table 3) (46). The results of (over)expressing the enzymes TDC, STR, and TDC and STR in C. roseus and other species are shown in more detail in Tables 3, 4, and 5, respectively.

A. Aspects of TIA Formation by Transgenic Cell Lines of C. roseus Overexpressing Tdc and Str

The highest ajmalicine production ever achieved in our laboratories was 77 mg L obtained after 16 days in a bioreactor under optimized conditions with a wild-type cell line of C. roseus (79). Such productivity is too low for commercialization; therefore, extensive studies were made to characterize genetically modified C. roseus cell cultures, in particular with regard to TIA formation, to investigate the effect of overexpression of the Tdc and Str genes on TIA formation and to follow their stability during long-term subculture. The available knowledge on wild-type cell cultures of C. roseus was the basis for these further studies with transgenic cell lines.

Transgenic cell lines were obtained from C. roseus seedling leaf cells by means of Agrobacterium tumefaciens-mediated transformation (Table 6). These cells overexpress the genes Str (S lines) and/or Tdc (T lines). The enzymes STR and TDC catalyze key steps in the biosynthesis of TIAs (Figs. 2 and 3). All our experiments were carried out using a two-stage production system in which biomass and product formation occur sequentially. Sufficient biomass was first obtained in growth medium (GM), and the cells were then inoculated into production medium (PM). Our PM does not contain phosphate, nitrate, or phytohormones, constituents that promote culture growth. Very little cell division therefore occurs in PM; most biomass increase is the result of starch biosynthesis (80).

As shown in Table 6, after the initiation of the transgenic calli lines we obtained 10 transgenic cell lines with six different T-DNA constructs (81). These transgenic cell cultures showed wide phenotypic diversity for TIA accumulation: non—TIA-accumulating and TIA-accumulating cell lines. After the initiation of transgenic cell suspension cultures from these calli lines, several cell lines gradually lost their viability after a number of subcultures. The surviving productive cell lines S10, SI (S lines, transgenic for STR), and T22 (T line, transgenic for TDC) were chosen for further studies of TIA accumulation. Both S and T lines showed higher STR and TDC activities, respectively, than the wild-type levels. The TIA accumulation profiles of the productive cell lines were as follows: line S10 produced ajmalicine, serpentine, catharanthine, and tabersonine and lines SI and T22 produced strictosidine, ajmalicine, serpentine, and catharanthine. Most notably, the viable transgenic cell lines showed many years of transgene stability.

Table 4 Studies with (Over)expression of the Strictosidine Synthase (Sir) Gene

Str (39-42) Origin of the clone Method Promoter Expressed in Results

Str (55) Origin of the clone Method Promoter Expressed in Observed as Results

Sir (71) Origin of the clone Method Promoter Expressed in Results

Rauvolfia serpentina (L.) Kurz (Apocynaceae) Bacuiovirus-based expression (42) Baculovirus polyhedrin (poth) (42)

E. coli (39,40), Saccharomyces cerevisiae (41), Spodoptera frugiperda (fall arm worm) (42) The clone has been identified (39).

Sir has been expressed in an enzymatically active form in E. coli. Addition of equimolar (15 mM) concentrations of secolo-ganin and tryptamine to the bacteria in either exponential or stationary phase resulted in the quantitative conversion of the precursors to strictosidine. Enzyme was in a soluble form within the bacterium but strictosidine accumulated in the medium (40). Expression of Str In S. frugiperda resulted in the overproduction of STR in a catalytically active form (max. 4 mg L ') (42).

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 70) Agrobacterium-mediated transformation using A. tumefaciens CAMV 35S

N. tabacum 'Xanthi' (Solanaceae) Transgenic plants

The transgenic plants had 3 to 22 times greater STR activity than C. roseus plants.

One difference In expression of STR between tobacco and C. roseus was the presence of a lower molecular weight in tobacco. Two distinct forms of the enzyme were produced in transgenic plants but only a single form was made in C. roseus plants. The second form of the protein is not a result of overexpression in tobacco but may represent differences in protein processing between tobacco and C. roseus.

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 70) Expression cassette polymerase chain reaction tac

E. coli

Induction of the Sir gene resulted in overexpression of the STR, which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant STR activity was about 30 nkat/mg.

Str (45) Origin of the clone Method Promoter Expressed in Observed as Str (43) Origin of the clone Expressed in

Results Str and Sgd (73) Origin of the clone Method Expressed in Results

C. roseus (L.) G. Don (Apocynaceae) (as in Ref. 72) Agrobacterium-medlaled transformation using A. tumefaciens CAMV 35S

Tabernaemontana pandacaqui Poir. (Apocynaceae) Transgenic plants

Rauvolfia serpentina Poir. (Apocynaceae) (as in Ref. 39)

22 bacterial strains (Aeromonas sp., Brochothrix thermosphacta, B. campestris, Bacillus lincheniformis, Cellulomonas uda, Citrobacter freundii, Enterobacter aerogenes, E. cloacae, Escherichia coli, E. coli T, Klebsiella oxytoca, K. pneumoniae, K. terrigena, Listeria grayi, L. innocus, Proteus mirabilis, P. vulgaris. Salmonella typhimurium, Serratia marcescens. Staphylococcus aureus, S. epidermidis, S. carnosus) Strictosidine was converted by the bacterial strains into vallesiachotamine and isovallesiachotamine.

C. roseus (L.) G. Don (Apocynaceae) Lithium acetate procedure Saccharomyces cerevisiae (yeast)

With secologanin and tryptamine feeding, transgenic yeast that was introduced with the genes Sfrand Sgd (strictosidine glucosidase) produced 2 g L"' strictosidine. Strictosidine is excreted into the medium and only small amounts of cathen-amine were found.

Growing the transgenic yeast on the sap obtained by pressing Symphoricarpus berries, which contain both sugar and secologanin, and feeding tryptamine, the system was able to produce large amounts of strictosidine (44).

Table 5 Studies with (Over)expression of the Tdc and Str Genes

Tdc and Str (74) Origin of the clone Method Promoter Expressed in

Observed as Results

Tdc and Str (75) Origin of the clone Method Promoter Expressed in Observed as Results

Tdc and Str (76) Origin of the clone Method Promoter Expressed in Observed as Results

C. roseus (L.) G. Don (Apocynaceae) (45) Agrobacterium-mediated transformation using A. rhizogenes CaMV 35S

Nicotiana tabacum 'Petit Havana' SR1 (Solanaceae), Lonicera xylosterum I. (Caprifoliaceae), Weigela 'Styriaca' (Diervilla-

ceae), C. pusillus 'Murray) G. Don (Apocynaceae), Cinchona officinalis 'Ledgeriana' (Rubiaceae) Transgenic hairy root cultures

In tobacco TDC and STR activities and tryptamine were detected.

High levels of secologanin (up to 1.25 mg g ' FW) was produced by L. xylosterum cultures.

The Weigela cultures after about 2 months turned brown and stopped growing.

Different TDC and STR activities between two hairy root cultures of C. pusillus were observed.

TDC and STR activities up to 4.1 and 450 pkat mgof protein, respectively, were recorded. High levels of tryptamine and strictosidine, up to 1.2 and 1.95 mg g~1 DW, respectively, were measured.

Agrobacterium-mediated transformation using A. tumefaciens CAMV 35S

Nicotiana tabacum 'Petit Havana' SR1 (Solanaceae) Transgenic cell suspension cultures

Transgenic tobacco cells showed relatively constant TDC and 2- to 6-fold greater STR activities compared with the non-

transformed tobacco cell in which the enzyme activities were not detectable. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin.

C. roseus (L.) G. Don (Apocynaceae) Agrobacterium-med\aied transformation using A. rhizogenes CaMV 35S

Cinchona officinalis 'Ledgeriana' (Rubiaceae) Transgenic hairy root culture

The products of TDC and STR, tryptamine and strictosidine were found in high amounts, 1.2 and 1.9 mg rrr' DW. However, no increase in desired quinoline alkaloids was observed compared with the amounts of 0.05 mg FW reported by Hamill et al. (77). One year later they lost the capacity to accumulate alkaloids.

C. roseus (L.) G. Don (Apocynaceae) {Tdc as in Ref. 51; Str as in Ref. 70) Particle bombardment CaMV 35S

Nicotiana tabacum 'Samsun' NN Transgenic plants

In 26% of the 150 Independent transgenic plants, both the plant Tdc and Str1 transgenes were silenced, 41 % demonstrated a preferential silencing of either transgene, 33% of them expressing both transgenes. Seven-week-old seedlings showed 24- and 110-fold variation in levels of TDC and STR1 activities, respectively.

C. roseus (L.) G. Don (Apocynaceae) Particle bombardment CaMV 35S

C. roseus (L.) G. Don (Apocynaceae) Transgenic calli cultures

Despite an increase in TDC and STR activities observed compared with the nontransformed cultures, TIA accumulation did not increase.

Origin of the clone Method Promoter Expressed in Observed as Results

Tdc and Str (Hilliou et al., personal communication) Origin of the clone Method Promoter Expressed in Observed as Results

Table 6 Gene Constructs and Derived Cultures

Number of established callus lines

Table 6 Gene Constructs and Derived Cultures

Number of established callus lines

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