Importance of L-DOPA
A hypofunction of the nigrostriatal and the mesolimbic dopaminergic systems appears to cause the symptoms of Parkinson’s disease (e.g. tremor, rigidity, akinesia). Post mortem studies have revealed a severe degradation of melanin-containing neurons in the substantia nigra zona compacta and a marked reduction of the contents of dopamine, its synthesizing enzymes and metabolites in various brain areas of Parkinsonian patients.
Both precursors in the biosynthesis of L-DOPA, i.e. L-tyrosine and 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA), may be taken up from the bloodstream by the brain. Dopamine is not able to pass the blood/liquor barrier. For this reason, L-DOPA has found a wide application for the symptomatic relief of Parkinson’s disease.
At present, commercially available L-DOPA is being synthesized chemically starting from vanillin. Due to its relatively great demand, a continuous search for alternative production possibilities is being carried out. Among these are the use of fungi for the bioconversion of N-formyltyrosine or N-carbobenzoxy-L-tyrosine, and the enzymatic coupling of DL-serine and pyrocatechol by tyrosine phenol lyase.
Occurrence of L-DOPA in Plants
The occurrence of L-DOPA in the seeds of Viciafaba has been known for a considerable time. Since then, this compound has been reported in plant parts of species of the legumes Baptisia, Lupinus, Mucuna (Stizolobium) and Vicia. Besides in Leguminosae, L-DOPA has also been demonstrated in the latex of Euphorbia lathyrus L. and in Aristolochia clematilis L..
Von Schantz et al. (1977) selected specimens of V. faba for a high L-DOPA content. The content of L-DOPA (as determined in the leaves) appeared to depend strongly on the stage of flowering of the plants, and increased from 0.13% (standard deviation 0.06) in the stage of bud formation through 0.34% (0.27%) and 0.30% (0.35%) in the stage of beginning and full flowering up to 0.68% (0.38%) when flowering is almost over. However, there was a considerable variation in the L-DOPA levels in different stadia, as is expressed by the standard deviations.
Of the Leguminosae, species of the genus Mucuna contained the highest levels of L-DOPA. This genus contains an estimated 160 species of scandent herbs or shrubs distributed in both the New and Old World tropics and sub-tropics. Some confusion appears to exist among taxonomists about the question whether Stizolobium species belong to the same genus as Mucuna, or whether both groups of species represent separate genera. However, the Index Kewensis considers the sections Stizolobium and Mucuna to be identical. Therefore, in this chapter species of Stizolobium and Mucuna are considered to belong to the same genus.
In 1920, Miller reported the presence of L-DOPA in the seeds of Stizolobium deeringiana, S. hassjoo and S. niveum. No quantitative determinations were reported. Almost two decades later, Damodaran and Ramaswami (1937) isolated L-DOPA from the seeds of M. pruriens in a yield of 1.5% (g/g). Since the application of L-DOPA for the symptomatic relief of Parkinson’s disease, a more intensive search for sources of L-DOPA has started.
Bell et al. (1971) isolated L-DOPA from the seeds of M. mutisiana in a 3.9% yield. They also isolated a possibly related metabolic compound, L-3-carb-oxy-6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline from these seeds. This compound was also reported to occur in the seeds of M. andreana, M. holtoni, M. pruriens, M. sloanei, M. urens and M. deeringiana (S. deeringiana),
Daxenbichler et al. (1972) isolated (-)-1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, which differs only in a methyl group from the previously mentioned compound, from the seeds of M. deeringiana.
Considerable amounts of L-DOPA were reported to occur in the seeds of different Mucuna species, in amounts of 3.1% -6.7% and 5.9%-9%.
Mucuna pruriens: Conclusions and Prospects
Based on the experiments performed by separate research groups, it can be concluded that L-DOPA is produced both by callus cultures and by cell suspension cultures of Mucuna (Stizolobium) species.
The productivity of the cell cultures is as high as several percent of the cell dry mass, which is quite noteworthy for plant cell cultures. The yields of the cell cultures compare favourably with those of the intact plant; in developing young plantlets the L-DOPA contents of the leaves, stems and roots rapidly declines below 0.1% of the dry mass upon maturation of the plant. Therefore, on the plant level the seeds of Mucuna plants appear to be the only promising source of L-DOPA. Because the seeds will only be a minor fraction of the total biomass of mature plants, exploitation for production of L-DOPA does not seem attractive.
The experiments performed with cell cultures clearly demonstrate that media suitable for propagative purposes have a different composition than those suitable for productive purposes. With the limited experiments described here, it is unlikely that the maximal productivity of the cell cultures by means of media alterations has been found. To gain a fundamental insight into the regulation phenomenon, experiments must be performed with continuous cultures, which allow the monitoring of productivity changes as a result of variation of single parameters.
A fundamental aspect of the mechanism of accumulation of L-DOPA in Mucuna cell cultures concerns the enzymatic regulation. The conversion of L-tyrosine into L-DOPA is performed by a phenoloxidase with a specific catecholase activity which is approximately 35-70 times higher than the specific cresolase activity. Compartmentation phenomena or the endogenous presence of a strong reductant may account for the accumulation of L-DOPA despite the presence of this phenoloxidase with a relatively high oxidizing activity.
Besides such a fundamental approach, another perspective may be found in the selection of high-producing cell lines. The experiments described in this article were carried out with “wild-type” cell lines. In this context, “wild-type” means that no selection of parent plants or cell lines was performed, nor was genetic variation intentionally generated. Selection and genetic engineering might eventually lead to a further optimization of the endogenous production of L-DOPA by cell suspension of M. pruriens. The selection of high producing cell lines has been applied successfully in a production process for shikonin by cell cultures of Lithospermum erythroryzon (). One needs, however, either a suitable marker (preferably a visual marker) or a very sensitive assay to perform selection experiments successfully.
For biotechnological purposes, in general cell suspension cultures are preferable to callus cultures, because of the higher growth rates and the easier control of the composition of the growth medium that can be obtained in cell suspension cultures, making the establishment of a continuous culture theoretically possible. In the experiments described here dry mass yields of the cell suspension cultures ranged between 14-21 mg/ml, while the L-DOPA content of these cells ranged from 0.2% -2% of the cell dry mass. From these figures, a concentration range of 0.14-2.13 mM (28-420 mg/1) for L-DOPA in the cell homogenates can be calculated. It seems needless to say, that considerable problems may arise in the down-stream processing of L-DOPA from such a complex cell homogenate, resulting in another significant cost factor.
Despite the high endogenous productivity of the cell suspension cultures of M. pruriens, it is questionable whether this production method will ever be exploited commercially. The intrinsic fermentation costs of plant cell cultures appear to be too high for this purpose. Productivity of the cell cultures will have to be at least 20% in reference to the cell dry weight, combined with a cell density of 30 g/1 (dry wt).
Another production possibility, however, lies in the application of alginate-en-trapped cells of M. pruriens for the bioconversion of L-tyrosine into L-DOPA, or the bioconversion of L-tyrosine derivatives in the corresponding catechols. When N-formyl-tyrosine is added to entrapped cells, N-formyl-DOPA can be produced very efficiently, resulting in a product yield of 119% in reference to cell dry weight. Another advantage of this production method is that the catecholic product is released for more than 95% in the cell-free medium, which greatly facilitates down-stream processing. Furthermore, due to the broad substrate specificity of the phenoloxidase involved, the entrapped cells can be used for the production of a whole range of catechols, among which compounds that hold great promise as alternative therapeutics in the treatment of Parkinson’s disease.
Selections from the book: “Medicinal and Aromatic Plants II”, 1989.