Biosynthesis, production and biological activity of singular betalain pigments

Resumen

Until now it was considered that the structural unit shared by all the betalains was betalamic acid, which is obtained after the spontaneous cyclization of 4,5-seco-DOPA, the resulting molecule after the activity of the enzyme 4,5-DOPA-extradiol-dioxygenase (DODA) on L-dihydroxyphenylalanine (L-DOPA). However, the results obtained in this Thesis shift this paradigm and show alternative sources of singular betalain pigments. The objectives of this Thesis were: 1. Searching for alternative sources of betalains in plants. 2. Physicochemical characterization of novel betalains and the study of their biosynthetic pathways. 3. Development of biotechnological processes for the production of betalains, with both microbiological and plant-based strategies. 4. Characterization of the health-promoting potential of singular betalains in the animal model Caenorhabditis elegans. In the search for novel sources of betalains, the pigments of Dionaea muscipula Ellis, also known as Venus flytrap, were investigated. The unambiguous establishment of anthocyanins in Dionaea muscipula Ellis supports the exclusion of the Droseraceae family from the order Caryophyllales from a phytochemical point of view and its inclusion in the order Nepenthales. The conditions for the development of Chenopodium quinoa callus lines on solid media that produce different betalain pigments have been established. The amount and variety of betalains present in the callus cultures indicate that the increase of the 6-benzylaminopurine hormone and the decrease of nitrogen in the culture medium are key factors to activate the biosynthetic pathway of betalains. The detection of two dopamine-derived betaxanthins in quinoa grains opened up the possibility of the existence of multiple dopamine-derived pigments in nature. In plants, they are probably hidden as minor peaks in HPLC recordings. A novel biosynthetic pathway was described, able to produce decarboxylated betalains in planta, as it has been demonstrated in C. quinoa. The key enzyme in the dopamine-derived pathway is a ring-cleaving 4,5-extradiol-dioxygenase that has been characterized at molecular and kinetic levels. The protein is a dual enzyme and expresses activity against the physiologically relevant substrates L-DOPA and dopamine. With the sole expression of the dual DODA enzyme, the profile of betalains naturally occurring in quinoa grains was reproduced biotechnologically. This is considered a neat demonstration of the simplicity of the square-shaped biosynthetic pathway needed to produce the pigments. An entire novel family of betalains - the 6-decarboxy-betalains - has been discovered, described and characterized. All members assayed present similar color properties and strong fluorescence. The twelve individual decarboxylated betalains studied in this Thesis present different behaviors in in vivo assays for lifespan and antioxidant capacity. The potential of 6-decarboxy-betalains as β-amyloid anti-aggregating molecules has also been studied. Four decarboxylated betalains have shown promising results with IC50 values in the micromolar range, of possible interest in the treatment or prevention of Alzheimer’s disease. Traditional carboxylated betalains and natural extracts reduce tumor growth in vivo and expand the lifespan of the Caenorhabditis elegans tumoral model (gld-1). The most effective molecule in the reduction of tumor size is L-tryptophan-betaxanthin. Betalains act not only as antioxidants, but also by regulating DAF-16 transcription factor. Betalains are revealed as potential anticancer phytochemicals, of possible importance in chemoprevention and treatment strategies. Novel betaxanthins have been designed from L-tryptophan-betaxanthin as a lead molecule. The novel pigments have been biotechnologically produced and purified, and their properties characterized. All the novel pigments show a significant reduction of the size of tumoral gonads in the tumoral animal model C. elegans (gld-1).