Exploring T-DNA gene mechanisms and optimizing centelloside production in Centella asiatica hairy root cultures
Year: 2023
Type: dissertation
Abstract: [eng] Plant biofactories constitute an eco-friendly alternative to chemical synthesis as they have a lower environmental impact and therefore contribute to a more sustainable future. Hairy root cultures, generated by infecting plant material with Rhizobium rhizogenes, are efficient plant biofactories for producing high-value bioactive plant compounds such as centellosides, which are found in Centella asiatica. An important challenge in plant biotechnology is the sustainable production of centellosides, which have widespread use in dermopharmaceuticals and cosmeceuticals. This PhD thesis aimed to investigate the effects of randomly integrated genes, specifically the rol genes, following R. rhizogenes infection in C. asiatica. The focus was on understanding the significance of changes in morphology and the expression of triterpene biosynthetic genes in hairy root lines to enhance centelloside production. Machine learning models were used to analyze the impact of T-DNA gene expression levels on morphological traits, hormonal profiles, and centelloside contents. The results indicated that a high expression of these genes was associated with increased centelloside production and positively influenced the expression of centelloside biosynthetic genes. Furthermore, we established connections between elevated centelloside levels and isopentenyladenosine, as well as between reduced centelloside content and abscisic acid. Proteomics analysis identified key biomarkers of centelloside production, notably ornithine cyclodeaminase. This biomarker is codified by the rolD gene, previously considered of low importance in hairy root induction, which was found to effect hairy root growth and centelloside content. To improve centelloside yields in high-growth C. asiatica hairy roots, various biotic elicitors were employed. Elicitation with coronatine/methyl jasmonate resulted in the highest total centelloside content, obtained at day 14 of treatment. Elicitation also notably enhanced the expression of centelloside pathway genes, particularly those involved in the final stages, leading to a greater centelloside production. To optimize centelloside yields in C. asiatica hairy roots through metabolic engineering, two transgenes were inserted: a transcription factor TSAR2 and squalene synthase from Arabidopsis thaliana. This resulted in the upregulation of genes involved in the initial steps of the centelloside metabolic pathway, thereby increasing the availability of precursors for an enhanced centelloside production. Some of these precursors were redirected toward phytosterol biosynthesis, leading to morphological changes in the hairy root phenotype and indicating a competitive interaction between the centelloside and phytosterol pathways.
Author: Alcalde Alvites, Miguel Ángel
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
Awards: G2660333250, G5805502524, G8776092940
OpenAlex ID: W6981753503