Production Of Terpenoid Indole Alkaloids Research Articles

Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus

Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.

Mevalonate kinase activity in Catharanthus roseus plants and suspension cultured cells

Mevalonate kinase is an early enzyme in plant isoprenoid biosynthesis. Its activity was studied in different parts of Catharanthus roseus (L.) G. Don (Apocynaceae) plants and in C. roseus suspension cultured cells. In the plant specific mevalonate kinase activities were found to be relatively high in the fruits, stem, roots, flowers and buds, and relatively low in young and completely elongated leaves. In suspension cultured cells, the specific mevalonate kinase activity increased during the exponential phase of growth, reaching a maximum of 0.5 nkat/mg protein at 4 days after subculturing. After 6 days, which corresponded to the beginning of the stationary phase, the specific activity decreased. After transferring 14-day-old C. roseus cells to a medium known to induce the production of terpenoid indole alkaloids, an increase in the specific mevalonate kinase activity was observed, reaching a maximum of 1.7 nkat/mg protein at 8 days after transfer. The alkaloid accumulation in the cultures was monitored, and ajmalicine was found to be the major product. Under standard conditions, mevalonate kinase activity showed a diurnal rhythm, with highest activities at 12:00 and 24:00.

Induction of a novel cytochrome P450 ( CYP96 family) in periwinkle ( Catharanthus roseus) cells induced for terpenoid indole alkaloid production

A full-length cDNA (designated CDS1) that encodes a cytochrome P450 enzyme (P450) was isolated by combining differential screening and asymmetric PCR amplifications of cDNA libraries from periwinkle ( Catharanthus roseus) cell cultures induced for terpenoid indole alkaloid (TIA) synthesis. The deduced polypeptide has 501 aminoacids, a heme-binding cystein domain (the P450 signature), residues conserved in most P450s, and a hydrophobic N-terminal region acting as membrane anchor. CDS1 was assigned as the first member of a new subfamily: CYP96C1. Hybridization of genomic periwinkle DNA with CYP96C1 probe suggested that the protein is encoded by a single-copy gene. CYP96C1 transcripts accumulated only in alkaloid-producing cells showing an association between the induction of the biosynthesis of TIAs and the expression of CYP96C1. The putative function of the CYP96C1 protein is discussed.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

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 in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium 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 a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc 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 the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

Comparison of terpenoid indole alkaloid production and degradation in two cell lines of Tabernaemontana divaricata

Two cell lines of Tabernaemontana divaricata derived from the same suspension culture accumulate different amounts of the terpenoid indole alkaloids O-acetylvallesamine and voaphylline. [(15)N]O-acetylvallesamine and [(15)N]voaphylline were added to the suspension cultures to investigate whether the lack of accumulating capacity of one of the cell lines was due to a low biosynthetic ability or to high turnover rates. The difference was shown to be due to the inability of the cell culture to biosynthesize both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred mainly during the stationary phase. The alkaloids added were chemically unstable under culture conditions. Under normal batch cell culture conditions chemical breakdown is thought to play a minor role in the total amount of compound transformed.