Biotechnological production of antioxidan petalains and evaluation of their functional capacity in the in vivo model Caenorhabditis elegans

Abstract

The antioxidant, anti-tumoral and anti-inflammatory properties of betalains have been mainly studied by employing extracts of plants of the order Caryophyllales. Betalains production and purification from edible parts of the plants have been limiting factors in the analysis of individual compounds. In addition, the lack of studies with pure, individual betalains has hindered the unequivocal confirmation of betalains as health-promoting compounds and the characterization of the most active structures. Thus, the objectives of this Thesis were: 1. Searching for alternative sources of betalamic acid-forming dioxygenases through enzyme-mining in non-native hosts. 2. Biotechnological production of individual betalains by heterologous expression of efficient DODA enzymes in bacterial cultures. 3. Construction and optimization of a machine for the automatic control of worms' lifespan and its application to the characterization of betalains. 4. Analysis of transcriptional changes in worms treated with betalains and subsequent biological confirmation by using mutant strains of C. elegans. The results obtained have shown that the synthesis of betalains is not restricted to plants. Microbial dioxygenases described in this Thesis present higher activity and affinity that those characterized from plants. Thanks to the superior activity of the DODA enzyme from Gluconacetobacter diazotrophicus, the early steps in the biosynthesis of betalains have been established for the first time. The cloning of this novel and efficient enzyme has led to the start-up of a biotechnological production system of individual pigments which improved the yield of previous methodologies. This novel biotechnological approach afforded a deeper study of the physical-chemical properties of betalains and the obtention of novel, unknown compounds such as chitosan-betaxanthin, the first fluorescent polymeric betaxanthin which might combine the fluorescent properties of betalains and the properties of chitosan, a sugar polymer widely used with medical purposes. In addition, the analysis of the properties of each betalain as bioactive compounds is now possible. These properties were analyzed using Caenorhabditis elegans, a small nematode widely used as animal model due to its fast, simple life cycle and its easy maintenance in the laboratory. Lifespan of C. elegans is a parameter widely used to describe the effect of different molecules in the aging of this animal model. With this purpose, Lifespan Machine, an automatic platform for the lifespan analysis was built. Thus, analysis of seventeen individual betalains showed that their consumption increases the lifespan of worms. The molecular mechanism underlying this effect was analyzed by using mutant strains and by performing microarray assays from the extraction of RNA from worms fed with betalains. The results showed that betalains modulate the expression of daf-16 or skn-1, orthologous genes to human FOXO and Nrf2, respectively. In both species, these genes are involved in pathways related to longevity and oxidative stress resistance and lead to the overexpression of hsp genes that encode HSP proteins involved in resistance to cancer and Alzheimer's. Therefore, the results obtained can open new lines of research in the search for effective treatments based on these molecules of plant origin. Parallel to the main topic, the device for the automatic analysis of C. elegans lifespan facilitates the study of other individual compounds, both natural and synthetic, in vivo in the animal model for the first time. Thus, the health-promoting effects of six flavonoids structurally related as well as the damage produced by the administration of six artificial food dyes were highlighted by using the Lifespan Machine and several techniques of microscopy. In addition, microarray analysis performed with worms treated with these compounds showed their target genes and how their consumption is able to modulate pathways related to longevity or oxidative stress.