Application of Priming Strategy for Enhanced Paclitaxel Biosynthesis in Taxus × Media Hairy Root Cultures

Abstract

Despite huge progress in biotechnological approaches to paclitaxel production, Taxus spp. in vitro culture productivity still remains a challenge. This could be solved by developing a new strategy engaging mechanisms of the primed defence response joined with subsequent elicitation treatment to circumvent limitations in paclitaxel biosynthesis. The hairy roots were primed by preincubation with β-aminobutyric acid (BABA) for 24 h or 1 week, and then elicited with methyl jasmonate (MeJA) or a mixture of MeJA, sodium nitroprusside and L-phenylalanine (MIX). The effect of priming was evaluated on a molecular level by examination of the expression profiles of the four genes involved in paclitaxel biosynthesis, i.e., TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), DBTNBT (3'-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) and PAM (phenylalanine aminomutase), as well as rolC (cytokinin-β-glucosidase), originated from the T-DNA of Agrobacterium rhizogenes. The maximum paclitaxel yield was achieved in cultures primed with BABA for 1 week and elicited with MIX (3179.9 ± 212 µg/g dry weight), which corresponded to the highest expression levels of TXS and BAPT genes. Although BABA itself induced the investigated gene expression over control level, it was not translated into paclitaxel production. Nevertheless, preincubation with BABA essentially affected paclitaxel yield, and the duration of BABA pretreatment seemed to have the most pronounced impact on its productivity.

Keywords: L-phenylalanine; elicitation; gene expression; methyl jasmonate; sodium nitroprusside; β-aminobutyric acid.

Figure 1

Fresh biomass increase of hairy roots cultivated under various culture conditions: control—untreated roots collected at day of inoculation (day “0”); BABA—roots treated with 100 µM β-aminobutyric acid (BABA) for 14 days; ETOH—roots treated with ETOH for 14 days without BABA, and variants: treated 24 h or 1 week with BABA with subsequent addition of ETOH for 14 days; MeJA—roots treated with MeJA for 14 days without BABA, other variants: treated 24 h or 1 week with BABA with prior to addition of MeJA for 14 days; MIX—roots cultivated for 14 days without BABA, and variants: treated 24 h or 1 week with BABA with subsequent addition of MIX of elicitors. Data represent mean values ± SD from three independent experiments. Data within groups denoted with the same letters are statistically significant (p ˂ 0.05).

Figure 2

Paclitaxel content [µg/g DW] determined in hairy roots cultivated in various culture conditions: (a) without BABA supplementation; (b) elicited after treatment with BABA for 24 h; (c) elicited after treatment with BABA for 1 week. Data represent mean values ± SD from three independent experiments. Data denoted with asterisks (*) are statistically significant (p ˂ 0.05).

Figure 2

Paclitaxel content [µg/g DW] determined in hairy roots cultivated in various culture conditions: (a) without BABA supplementation; (b) elicited after treatment with BABA for 24 h; (c) elicited after treatment with BABA for 1 week. Data represent mean values ± SD from three independent experiments. Data denoted with asterisks (*) are statistically significant (p ˂ 0.05).

Figure 3

The gene expression profiles in control (a) and in 100 µM BABA-treated (b) hairy roots. Data represent mean values ± SD from three independent experiments.

Figure 4

The effect of elicitation on relative gene expression profiles in hairy root cultures treated with ETOH (a), MeJA(b) or MIX (c). Data represent mean values ± SD from three independent experiments.

Figure 4

The effect of elicitation on relative gene expression profiles in hairy root cultures treated with ETOH (a), MeJA(b) or MIX (c). Data represent mean values ± SD from three independent experiments.

Figure 5

The effect of BABA24 treatment followed by ETOH (a), MeJA (b) or MIX (c). Elicitation on relative gene expression profiles in hairy root cultures. Data represent mean values ± SD from three independent experiments.

Figure 5

The effect of BABA24 treatment followed by ETOH (a), MeJA (b) or MIX (c). Elicitation on relative gene expression profiles in hairy root cultures. Data represent mean values ± SD from three independent experiments.

Figure 6

The effect of BABA1 treatment followed by ETOH (a), MeJA (b) or MIX (c) elicitation on relative gene expression profiles in hairy root cultures. Data represent mean values ± SD from three independent experiments.

Figure 6

The effect of BABA1 treatment followed by ETOH (a), MeJA (b) or MIX (c) elicitation on relative gene expression profiles in hairy root cultures. Data represent mean values ± SD from three independent experiments.