Mercedes Bonfill

Summary Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin‐aminophenylpropanoyl‐13‐ O ‐transferase (BAPT) and 3′‐ N ‐debenzoyltaxol N ‐benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux‐limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4‐mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two‐stage system and stimulated for taxane production by a dual elicitation treatment with 1 μ m coronatine plus 50 m m of randomly methylated‐β‐cyclodextrins. A high overexpression of BAPT (59.72‐fold higher at 48 h) and DBTNBT (61.93‐fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS , TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT ‐overexpressing line.

Centella asiatica is a medicinal plant with a rich tradition of use for its therapeutic properties. Among its bioactive compounds are centellosides, a group of triterpenoid secondary metabolites whose potent pharmacological activities have attracted significant attention. Metabolic engineering has emerged as a powerful biotechnological tool to enhance the production of target compounds. In this study, we explored the effects of overexpressing the squalene synthase (SQS) gene and transcription factor TSAR2 on various aspects of C. asiatica hairy root lines: the expression level of centelloside biosynthetic genes, morphological traits, as well as squalene, phytosterol, and centelloside content. Three distinct categories of transformed lines were obtained: LS, harboring At-SQS; LT, overexpressing TSAR2; and LST, simultaneously carrying both transgenes. These lines displayed noticeable alterations in morphological traits, including changes in branching rate and biomass production. Furthermore, we observed that the expression of T-DNA genes, particularly aux2 and rolC genes, significantly modulated the expression of pivotal genes involved in centelloside biosynthesis. Notably, the LS lines boasted an elevated centelloside content but concurrently displayed reduced phytosterol content, a finding that underscores the intriguing antagonistic relationship between phytosterol and triterpene pathways. Additionally, the inverse correlation between the centelloside content and morphological growth values observed in LS lines was countered by the action of TSAR2 in the LST and LT lines. This difference could be attributed to the simultaneous increase in the phytosterol content in the TSAR2-expressing lines, as these compounds are closely linked to root development. Overall, these discoveries offer valuable information for the biotechnological application of C. asiatica hairy roots and their potential to increase centelloside production.