Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel

Abstract

SummaryPaclitaxel 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 μm coronatine plus 50 mm 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.

Effects of Different Doses of sUV-B Exposure on Taxane Compounds’ Metabolism in Taxus wallichiana var. Mairei

“…Mairei, and its metabolism was regulated by many metabolic enzymes. It has been reported [35] that increasing the expression level of the Ts gene facilitates the promotion of paclitaxel accumulation. Ts was thought to be a key enzyme gene that catalyzes paclitaxel synthesis [36].…”

Regulatory microRNAs and phasiRNAs of paclitaxel biosynthesis in Taxus chinensis

“…Various approaches, including chemical synthesis, semi-chemical synthesis, plant tissue/cell culture, endophytic fungal synthesis and others, have been explored to develop sustainable methods for paclitaxel production ( Fett-Neto et al., 1992 ; Balogu and Kinston, 1999 ). Many genes in the paclitaxel biosynthetic pathway were also identified, which can be used to screen bottleneck enzymes to optimize metabolic engineering for paclitaxel production ( Howat et al., 2014 ; Kuang et al., 2019 ; Perez-Matas et al., 2024 ). The biosynthesis of paclitaxel involves at least 19 steps, from diterpene precursor geranylgeranyl diphosphate (GGPP) to the final product ( Hefner et al., 1998 ; Jarchow-Choy et al., 2014 ; Liao et al., 2016 ).…”