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

Abstract

Cell suspension and root cultures of Peganum harmala were established expressing a tryptophan decarboxylase cDNA clone from Catharanthus 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 feeding L-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 13-carboline alkaloids, the other tryptamine-derived metabolites of P. 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.

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

“…A cDNA clone encoding TDC was isolated from C. roseus (DeLuca et al, 1989) and subsequently its corresponding gene . The regulation of tdc expression was studied in C. roseus Pasquali et al, 1992) and Nicotiana tabacum and its overexpression in N. tabacum (Songstad et al, 1990;Goddijn et al, 1993;Poulsen et al, 1994) and Peganum harmala (Berlin et al, 1993). A number of cDNA clones encoding SSS were isolated from Rauvolfia serpentina (Kutchan et al, 1988) and C. roseus (McKnight et al, 1990;Pasquali et al, 1992) and expression studies were carried out in C. roseus plants and cell suspensions (Pasquali et al, 1992;Roewer et al, 1992).…”

Metabolic Engineering of Plants for Alkaloid Production

“…TDC has, however, proved useful in other systems like canola as a means of diverting flux to a metabolic sink to reduce undesired products (Chavadej et al, 1994). It has also been used to manipulate the alkaloid contents of Cinchona officinalis (Geerlings et al, 1999) and Peganum harmala (Berlin et al, 1993). With the additional genes now available, more metabolic engineering studies should be pursued.…”

Beyond a neurotransmitter: The role of serotonin in plants

“…Similarly, plants first synthesize tryptophan via the chorismate pathway which is then decarboxylated by tryptophan decarboxylase (TDC) to form tryptamine, another bioactive amino acid and common secondary metabolite precursor. Tryptamine is then hydroxylated by tryptamine-5-hydroxylase (T-5-H) to form serotonin ( Figure 1) [9,15,16] . In plants, TDC is the rate limiting enzyme which controls flux through the pathway.…”

“…Serotonin was first found in the medicinal herb Mucuna pruriens (L.) DC (cowhage) in the 1950's [8] . Though interest in phytoserotonin was primarily focused on efforts to enhance production in several plant species in hopes of providing source materials for pharmaceutically relevant compounds [9][10][11] , interest in the role of serotonin in plants has slowly followed, and was greatly encouraged and supported by the discovery of its biosynthetic product melatonin in plants in the mid 1990's [12,13] . This review provides a brief overview of the roles, mechanisms and functions of these important neurotransmitters in the plant system.…”