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

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 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.

Introduction

Paclitaxel, commercialized as Taxol® or Abraxane®, is a chemotherapeutic diterpene alkaloid used to treat various types of cancer, such as breast, ovarian and squamous cancers (Hanano et al., 2022). It is produced by the plant genus Taxus, with the highest amounts found in T. chinensis, T. brevifolia and T. baccata (Xiong et al., 2021). However, its scarcity in nature (it is only found at concentrations lower than 0.05% within the plant) and the complex paclitaxel biosynthetic pathway that makes chemical synthesis non‐commercially viable, forced to explore biotechnological alternatives for the production of this compound (Howat et al., 2014). In this sense, the use of Taxus spp. cell suspension cultures represents the current method of choice to obtain paclitaxel, owing to the controlled conditions and cost‐effectivity of these biotechnological systems. Despite this, the production levels remain insufficient to satisfy the market demand (Mutanda et al., 2021).

Concerning paclitaxel biosynthetic pathway, it is a very complex process and comprises 19 steps, involving eight oxidation steps, five acetyl/aroyl transferase steps, a C4β, C20‐epoxidation reaction, a phenylalanine aminomutase step, an N‐benzoylation and two CoA esterifications (Figure 1). Despite the pathway has been deeply studied, only 14 steps have been fully characterized to date (Sanchez‐Munoz et al., 2020). Biosynthesis starts with the condensation of three isoprenyl diphosphate (IPP) units with one dimethylallyl diphosphate (DMAPP) to form geranylgeranyl diphosphate (GGPP) by geranylgeranyl diphosphate synthase (GGPPS). This compound is cycled to form taxa‐4(5),11(12)‐diene by taxadiene synthase (TXS) forming the taxane skeleton. Then, after several region‐ and stereospecific chemical modifications involving cytochrome‐P450 hydroxylases, oxidases and acyl transferases the intermediate baccatin III is obtained by the enzyme DBAT (10‐deacetylbaccatin III 10‐O‐acetyltransferase). Finally, the last steps include the binding of the side chain by the enzyme BAPT (baccatin III 13‐O‐(3‐amino‐3‐phenyl‐propanoyl) transferase), the hydroxylation of the side chain at carbon 2′ and the benzoylation step by DBTNBT (3′‐N‐debenzoyl‐2′‐deoxytaxol‐N‐benzoyl transferase), which forms the end product paclitaxel (Howat et al., 2014).

Figure 1.

Paclitaxel biosynthetic pathway summary. Enzymes selected for the study of gene expression appear in yellow boxes. Besides, the target genes for metabolic engineering experiments, BAPT and DBTNBT, are in red circles. Important itermediate compounds of the biosynthetic pathway are indicated by yellow circles. Enzyme abbreviations: BAPT, 13‐O‐(3‐amino‐3‐phenylpropanoyl)‐CoA transferase; DBAT, 10‐deacetylbaccatin III‐10‐O‐acetyltransferase; DBTNBT, 3′‐N‐debenzoyl‐2′‐deoxytaxol‐N‐benzoyltransferase; GGPPS, geranygeranyl diphosphate synthase; PAM, phenylalanine aminomutase; T10βOH, taxane‐10β‐hydroxylase; T13αOH, taxane‐13α‐hydroxylase; T14βOH, taxane‐14β‐hydroxylase; T1βΟH, taxane‐1β hydroxylase; T2′αOH, taxane 2′α‐hydroxylase; T2αOH, taxane‐2α‐hydroxylase; T5αOH, taxane‐5α‐hydroxylase; T7βOH, taxane‐7β‐hydroxylase; T9αΟH, taxane‐9α‐hydroxylase; TAT, taxane‐5α‐ol‐O‐acetyltransferase; TBT, taxane‐2α‐O‐benzoyl transferase; TXS, taxadiene synthase. Figure adapted from Palazon et al. (2003).

Metabolic engineering has been successfully used to increase the production of specialized metabolites in organisms such as bacteria, yeast and plant cells. In this approach, multiple techniques are applied to enhance the cellular metabolism of the compounds of interest by modifying the genetic material of the organism (Li et al., 2015). Three main strategies have been developed: pathway overexpression, transcription factor engineering and knockdown/knockout of competing pathways. Whereas the latter technique aims to eliminate undesired metabolic pathways that could compete for precursors of the target compound, transcription factors are modified to regulate gene expression in a particular metabolic pathway. Finally, rate‐influencing genes are overexpressed to increase the efficiency of bottleneck enzymes in the biosynthetic pathway of a desired compound (Pickens et al., 2011). Although the paclitaxel biosynthetic pathway has still not been completely elucidated, the main flux‐limiting steps have been determined and consequently, pathway overexpression has emerged as the state‐of‐the art approach in metabolic engineering of Taxus cell cultures (Sanchez‐Munoz et al., 2020). Among the achievements to date, studies have identified that the limiting steps of the paclitaxel biosynthetic pathway are catalysed by the downstream enzymes BAPT and DBTNBT, which showed very little variation in their gene expression under elicitation conditions (Kashani et al., 2018).

Overexpression of a plant biosynthetic pathway involves the insertion of additional DNA into the genome, a process commonly achieved using a vector containing the gene of interest under the regulation of a strong promoter, such as the widely used cauliflower mosaic virus 35S promoter (CaMV35S; Brzycki Young and Roberts, 2021). Traditionally, plant genetic transformation has been performed using species of the Gram‐negative bacterial genus Rhizobium (formerly Agrobacterium) as vectors, namely R. radiobacter and R. rhizogenes, which harbour the pTi or pRi plasmids, respectively (Gelvin, 2003).

Although numerous attempts have been made to obtain transformed cells (via R. radiobacter) or hairy root cultures (via R. rhizogenes) of Taxus, the difficulty of genetically transforming this gymnosperm genus, together with low transformation efficiency and slow in vitro recovery, means that few have been successful (Perez‐Matas et al., 2023). Briefly, Furmanowa and Syklowska‐Baranek (2000) succeeded in establishing transformed hairy root cultures of T. x media by direct infection of explants with three different strains of R. rhizogenes. However, the root cultures took a long time to appear after infection (19 weeks) and their growth rate during the first one and a half years was slow. Once a stable culture was achieved, elicitation experiments with methyl jasmonate (MeJA) demonstrated that the paclitaxel production of the transformed roots was threefold higher compared to the control roots. The same research group observed that precursor feeding (L‐phenylalanine and p‐aminobenzoic acid) together with MeJA increased paclitaxel production 14‐fold compared to untreated root cultures (Syklowska‐Baranek et al., 2009). Additionally, Kim et al. (2009) induced the formation of hairy roots after infecting T. cuspidata seedlings with three wild strains of R. rhizogenes, comparing a direct infection method with liquid or solid co‐culture. After 10 months of subculture, three stable hairy root lines were obtained and after the best line was selected, elicitation experiments with MeJA resulted in a paclitaxel yield of 52.6 mg/L. Exposito et al. (2010) managed to establish T. x media hairy root cultures overexpressing the T. baccata TXS gene (taxadiene synthase) under the control of the CaMV35S promoter by direct infection with the R. rhizogenes LBA 9402 strain or R. radiobacter C58C1 carrying the A. rhizogenes RiA4 plasmid and the binary plasmid pCA‐TXS‐His. Despite this achievement, the hairy roots obtained showed poor growth and were dedifferentiated to generate T. × media cell cultures. Syklowska‐Baranek et al. (2015) substantially improved the growth and taxane production of T. × media transgenic roots overexpressing the taxadiene synthase gene (TXS) by growing them in two‐phase liquid cultures with aerated or degassed perfluorodecalin (PFD) together with 100 μm MeJA. More recently, Chang et al. (2017) induced T. sumatrana crown galls and hairy roots by infecting stem and leaf segments with R. radiobacter strain A281 and R. rhizogenes strains AR1600 and ATCC15834. Considerable variation in the transformation efficiencies was observed among the different Rhizobium strains and T. sumatrana clones. Finally, He et al. (2023) has been the only research group to date to obtain hairy roots from ex vitro seedlings of T. baccata by transformation with the wild type R. rhizogenes A4 strain, although the roots were not acclimatized to in vitro conditions and the production of paclitaxel was not tested.

The aim of the present research was to enhance paclitaxel production in T. baccata in vitro cultures by overexpressing the two flux‐limiting genes in the downstream taxane biosynthetic pathway, BAPT and DBTNBT, through a R. rhizogenes A4‐mediated transformation. The engineered T. baccata hairy roots were dedifferentiated to obtain callus cultures, which were elicited with 1 μm coronatine and 50 mm of randomly methylated‐β‐cyclodextrins (β‐CDs), and the impact of BAPT and DBTNBT gene overexpression on the production of cell‐associated and extracellular paclitaxel and related taxanes was analysed. Additionally, the transcriptional profiles of key genes in the paclitaxel biosynthetic pathway were determined: GGPPS (geranylgeranyl diphosphate synthase); TXS (taxadiene synthase); DBAT (10‐deacetylbaccatin III 10‐O‐acetyltransferase); T7βOH (taxane 7‐β‐hydroxylase); BAPT, DBTNBT and PAM (phenylalanine aminomutase).

Results

Transformation of Taxus baccata seedlings for hairy root and callus induction

Embryos of T. baccata sterilized seeds were grown in hormone‐free modified DCR medium to generate the seedlings (Figure 2a,b). For hairy root induction, a total of eighty 8‐week‐old seedlings were directly infected with a mixture of the two A4 R. rhizogenes strains harbouring the plasmids pK7WG2‐BAPT and pCAMBIA_1301‐DBTNBT. After 9–11 months, eight root lines (L1, 4, 6, 7, 8, 9, 10 and 11) were obtained either directly from the site of infection or from nodular calli generated at the wound, resulting in a root induction frequency of 10%. The 2–3‐cm‐long roots were excised and cultured individually in solid hormone‐free modified DCR medium and subcultured biweekly. During the first months after induction, only 50% of the roots (L4, 9, 10 and 11) exhibited a stable but slow growth and branching (Figure 2c,d), and callus formation was observed occasionally.

Figure 2.

Steps for the induction and establishment of Taxus baccata hairy root and callus cultures. (a) T. baccata seeds used for embryo culture; (b) 8‐week‐old seedlings grown in hormone‐free modified DCR medium and used for bacterial infection; (c) newly induced hairy roots emerging after 9–11 months of infection; (d) 8‐week‐old hairy roots; (e) stable 1‐year‐old hairy root line confirmed by PCR; (f) 5–7 cm‐long sections of established hairy roots after 2 weeks in callus induction medium; (g) hairy root line surrounded by overgrown callus after 4 weeks in induction medium; (h) stable transgenic callus line.

To overcome the growth limitations of the established root lines, 5–7‐cm‐long sections were excised and placed in callus induction medium (see Biomass production and viability of T. baccata cell lines). After 2–4 weeks, emerging callus tissues were separated from the roots and cultured in Gamborg's B5 growth medium for biomass production.

Molecular characterization of the Taxus root and derived callus lines

PCR analysis of the four stable root lines selected for their growth capacity (L4, 9, 10 and 11) and their derived callus cultures revealed the integration of the rolC gene only in L9 (Figure S1a), confirming its genetic transformation by R. rhizogenes. In the callus cultures derived from the slower‐growing root lines (L1, 6, 7, 8), PCR analysis detected the rolC gene only in L6 (Figure S1b). The virD gene was not detected in any root line or derived calli (Figure S1b,c), indicating successful elimination of the bacteria.

In the rolC‐transformed L9 and L6, the integration of the bacterial genes rolA, rolB and aux1 was also verified (Figure S1d,e), further confirming their transformed nature.

These results demonstrate the low efficiency of Taxus transformation. After a low rate of root formation (10%), only 25% of the root lines tested were transformed and only one was sufficiently stable to be cultured. Thus, the success rate of the entire infection process was a mere 2.5%. Furthermore, after 1 year of culture, the stable root cultures showed the typical hairy root phenotype only on the root tips (Figure S2), and the transformed roots (L9) resembled the untransformed control.

The main objective of this study was to alleviate the taxane metabolic bottleneck by overexpressing the flux‐limiting genes of the taxane biosynthetic pathway, BAPT and DBTNBT, both under the control of one copy of the constitutive CaMV35S promoter. Genomic PCR amplification revealed clear bands of 600 bp for 35S‐DBTNBT in L9 roots and calli, indicating the successful integration of the extra copy of the DBTNBT gene from the plasmid pCAMBIA_1301‐ DBTNBT (Figure S3a). In contrast, the extra copy of the BAPT gene was not integrated in L9, as only bands corresponding to the wild type gene, including introns, were amplified (>419 bp), differing in size from the cDNA of the bacterial plasmid pK7WG2‐BAPT (419 bp; Figure S3b). In L6 roots and calli, the inserted BAPT gene was confirmed by PCR amplification of the 680 bp band for 35S‐BAPT, which was absent in the wild type roots (Figure S3c).

Genetic integration of the T‐DNA of both plasmids was also confirmed by the detection of the antibiotic resistance genes (Figure S4a,b).

Based on these results, T. baccata lines L6 and L9 are referred to henceforth as the BAPT line and DBTNBT line, respectively. Additionally, L4 was selected as a wild type (untransformed) line for further experiments and is referred to as the WT line.

Biomass and taxane production of T. baccata root cultures

Root lines able to produce enough biomass were selected for further experiments: the DBTNBT and WT lines. The biomass production of both lines expressed as fresh weight increase (FWI) and fresh weight under elicitation (FEW) reflected the slow growth of the WT and DBTNBT root lines throughout the experiment (Figure S5). On average, the FWI index reached a value of approximately 1.2, without significant differences between the two lines. The FWE values were slightly lower, suggesting that elicitation reduced the FW, especially in the DBTNBT line, which experienced a 26% growth reduction at the end of the culture.

The DBTNBT and WT root lines were also analysed for total taxane production, quantified as the sum of 10‐deacetylbaccatin III (DABIII), baccatin III (BACIII), 10‐deacetyl taxol (DAT), cephalomannine (CEPH) and paclitaxel (PTX), which was determined at day 0 (after 28 days of culture in control conditions) and day 14 post‐elicitation. Although only side chain‐bearing taxanes were detected (CEPH, DAT and PTX), clear differences were observed between the two lines (Figure S6a). At day 0, 86.73% of the total taxane content in the WT line corresponded to CEPH (1.64 mg/L) and the rest to PTX (0.25 mg/L), whereas in the DBTNBT line, only PTX was found. At day 14, 96.86% of the total taxane content in the control WT line consisted of DAT (17.97 mg/L), whereas in elicited samples CEPH predominated (46.22%), followed by DAT (33.22%) and PTX (20.56%). The only taxanes found in control and elicited DBTNBT root cultures were CEPH and PTX, although in different proportions. In control conditions, most of the taxane content (78.77%) was CEPH (6.38 mg/L), whereas after the dual elicitation treatment the PTX content represented circa 70% of the total (3.99 mg/L). Finally, concerning the extracellular (Figure S6b) and intracellular (Figure S6c) taxane distribution, in all root lines the vast majority of the CEPH produced was found inside the roots, while DAT was predominantly extracellular (80%–90%). The distribution of PTX varied according to the culture conditions, being mainly intracellular in the control (83.87% in WT and 100% in DBTNBT lines), and extracellular in elicited cultures (89.42% in WT and 78.07% in DBTNBT lines).

Given the slowness of growth and taxane production in the biotechnological platform based on the T. baccata root system, the roots were dedifferentiated to establish callus lines and cell suspensions.

Biomass production and viability of T. baccata cell lines

For biomass determination, the root‐derived cell suspensions were maintained for 12 days in the growth medium (GM), after which the biomass was transferred to the production medium (PM) (inoculum of 300 g/L), which stimulates specialized metabolism in detriment of cell growth. For the experiments, BAPT, DBTNBT and WT cell lines were grown in control (C) and elicited (E) conditions for 24 days. The culture was proceeded with the same elicitation treatments as for the roots but without precursor feeding or priming with BABA.

The growth curves, expressed as FW (Figure 3a), indicate that the control WT line had the highest growth during most of the experiment, its biomass steadily increasing until day 20 (362.11 g/L), after which it decreased by 8.71%. The control DBTNBT line, despite an initial decrease, subsequently reached the maximum FW of the entire study at day 24 (387.33 g/L). The control BAPT line presented a similar growth pattern but with a lower final FW (329.14 g/L). The effect of elicitation was most evident in the DBTNBT line, followed by the WT line, both presenting the lowest FW values, mainly in the last two time points. Growth curves expressed as DW (Figure 3b) exhibited similar trends to those of FW. Besides, cell viability was maintained in all lines at values >70% and only showed slight differences between control and elicited conditions at days 8 and 16 of culture (Figure 4).

Figure 3.

Fresh weight (FW) (a) and dry weight (DW) (b) growth curves of three Taxus baccata cell lines in elicited (_E) and control conditions (_C): WT, wild type line; DBTNBT, a 3′‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing line; and BAPT, a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing line. Elicitation was performed with 1 μm coronatine +50 mm β‐methyl‐cyclodextrins. Values presented are means ± SD (n = 3). Values followed by different asterisk symbols are significantly different (P ≤ 0.05) according to Tukey's honestly significant difference test.

Figure 4.

Cell viability study (% of living cells) of three Taxus baccata cell lines in elicited (_E) and control conditions (_C): WT, wild type line; DBTNBT, a 3′‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing line; and BAPT, a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing line. Elicitation was performed with 1 μm coronatine +50 mm β‐methyl‐cyclodextrins. Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey's honestly significant difference test.

Taxane production of T. baccata cell lines

Total taxane production was determined in the DBTNBT, BAPT and WT lines in both control and elicited conditions, and expressed as the sum of DABIII, BACIII, DAT, CEPH and PTX, as explained in Biomass and taxane production of T. baccata root cultures.

The highest taxane content was obtained in the elicited DBTNBT line, where production also peaked earlier (Figure 5), reaching 367 mg/L at day 8 of culture, which was 9.91‐fold and 5.47‐fold higher than in elicited BAPT (37.21 mg/L) and WT (67.52 mg/L) lines, respectively, at the same sampling day. In contrast, the maximum taxane yield of BAPT (181.56 mg/L) and WT lines (71.01 mg/L) was found at day 16, when that of the DBTNBT line (203.27 mg/L) was still 1.12‐fold and 2.86‐fold higher, respectively. In all lines, the total taxane content decreased gradually from days 16 to 24 to 123.24, 70.71 and 37.06 mg/L in DBTNBT, BAPT and WT lines, respectively. In control conditions, significant differences between lines were only observed at days 8 and 16 of culture. At day 8, the BAPT line reached a value of 64.27 mg/L, which was 3.32‐fold and 9.29‐fold higher than in DBTNBT and WT lines, respectively. At day 16, the content in the unelicited DBTNBT line peaked at 18.44 mg/L, compared to 12.12 and 8.44 mg/L in WT and BAPT lines, respectively.

Figure 5.

Total taxane contents accumulated throughout the experiment in three Taxus baccata cell lines in elicited (_E) and control conditions (_C): WT, wild type line; DBTNBT, a 3′‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing line; and BAPT, a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing line. Elicitation was performed with 1 μm coronatine +50 mm β‐methyl‐cyclodextrins. Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey's honestly significant difference test.

The taxane excretion rates (Figure S7) indicated that in control conditions most taxanes remained inside the producer cells in all the analysed lines. In the BAPT line at the beginning of the experiment, circa 50% of the taxanes were intracellular, compared to 70% and 80% in WT and DBTNBT lines, respectively. In the WT line, intracellular taxanes always remained circa 70%, except at day 20 (53.62%), whereas in the BAPT line, the amount was always above 50%, except at day 8, and in the DBTNBT line, about 70%, except at day 12 (34.78%). However, after treatment with COR plus β‐CDs, the amount of taxanes excreted into the medium increased significantly in all lines (an average of 71%, 78% and 81% for WT, BAPT and DBTNBT lines, respectively). Interestingly, the lowest proportion of extracellular taxanes was found at day 24 (the end of the experiment) in all lines (59.40%, 70.16% and 75.91% for the WT, DBTNBT and BAPT lines, respectively), whereas the highest varied from day 8 in the DBTNBT line (91.16%), day 12 in the BAPT line (84.82%), to day 16 in the WT line (79.75%). These results indicate that DBTNBT line exhibited the highest production of secreted taxanes to the culture medium.

The total contents and partitioning of individual taxanes was also studied in the cell suspensions. As shown in Figure 6a, in the control WT line, PTX was the most abundant individual taxane at days 12 and 16 (6.22 and 8.09 mg/L, respectively), but by the end of the experiment, it was overtaken by BACIII and CEPH, which peaked at day 20 (5.26 and 3.87 mg/L, respectively). Elicitation of the WT line induced a significant increment in the PTX contents, especially at day 16 (53.83 mg/L) when it had increased 6.65‐fold. Interestingly, the treatment also stimulated the synthesis of DAT, DABIII and BACIII.

Figure 6.

Total contents of the individual taxanes studied (DABIII, BACIII, DAT, CEPH and PTX) throughout the experiment in the three Taxus baccata cell lines in elicited (_E) and control conditions (_C): WT (a), wild type line; DBTNBT (b), a 3′‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing line; and BAPT (c), a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing line. Elicitation was performed with 1 μm coronatine +50 mm β‐methyl‐cyclodextrins. Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey's honestly significant difference test.

In the DBTNBT cell line (Figure 6b), PTX and CEPH were the most abundant taxanes throughout the experiment in control conditions. PTX levels decreased gradually from 8.76 mg/L at day 8 to 6.48 mg/L at day 16, while those of CEPH remained relatively high, experimenting a sharp earlier decline at day 12 (0.99 mg/L). DAT was not detected in this line in control conditions. As in the WT line, elicitation significantly increased PTX levels to 100 mg/L or more throughout the experiment, reaching a maximum at day 8 (308.15 mg/L), which was 35.19‐fold higher compared to the control at the same time point. At the end of the experiment, PTX contents declined gradually to 97.66 mg/L at day 24. DAT levels were also clearly stimulated by elicitation, peaking at day 8 (32.70 mg/L). Although elicitation showed no effects on DABIII, it clearly increased the content of BACIII (9.87‐fold at day 8) and CEPH (5.59‐fold at day 24).

In the BAPT line (Figure 6c), the most abundant taxane in control conditions was DAT, which exhibited two production peaks, at days 8 (58.94 mg/L) and 16 (3.055 mg/L), whereas CEPH, DABIII and BACIII contents remained relatively constant throughout the experiment. PTX levels were very low and peaked at day 8 (1.33 mg/L). Nevertheless, the elicitation strategy significantly increased PTX contents at all time points, with a maximum at day 16 (134.01 mg/L), when it was 249.08‐fold higher compared to the control. Under these conditions, PTX represented circa 60%–70% of the total taxane content during the experiment. Elicitation also increased the levels of the other taxanes, but to a much lesser extent; despite doubling, CEPH, DABIII and BACIII levels remained relatively constant.

The extracellular and intracellular partitioning of individual taxanes (Figures S8–S10) was similar to that observed for total taxanes. Accordingly, with the exception of DAT and BACIII in the DBTNBT line (Figure S9), taxane excretion to the medium was clearly increased by the addition of β‐CDs plus COR, especially PTX, DAT and BACIII. CEPH was the only taxane that remained almost completely intracellular in both control and elicited conditions.

Transcript profile of taxane biosynthetic genes in cell lines

The expression levels of key taxane biosynthetic genes were assessed by RT‐qPCR and their relationship with the pattern of taxane production was studied. Expression was determined from 1 h to 3 days after the dual elicitation treatment and expressed referring to the levels in cell suspensions at day 12 of culture in the optimal GM.

Besides the two flux‐limiting genes BAPT and DBTNBT, the target genes were TXS, encoding taxadiene synthase, T7βOH, encoding taxadiene 7‐β‐hydroxylase and DBAT, encoding 10‐deacetylbaccatin III 10‐O‐acetyltransferase, which act in the beginning, middle and last steps of the taxane pathway, respectively. Also determined were the expression levels of the PAM gene, which is involved in the formation of the C‐13 side chain that converts α‐phenylalanine to β‐phenylalanine, and the GGPPS gene, which is crucial for the formation of GGPP, the universal precursor of all diterpenoids.

GGPPS transcript levels were high in both transgenic lines under control and elicited conditions (Figure 7a), but the expression pattern differed. In the DBTNBT line, a gradual increase in transcripts was observed, although in control conditions maximum expression was reached earlier (at 12 h) than in the elicited line (at 48 h), and was also much lower (26.56‐fold vs. 46‐fold increase, respectively). In the BAPT line, there were several peaks of GGPPS gene expression instead of a gradual increase, with the maximum value found in elicited cultures at 48 h (61.57‐fold increase) compared to 6 h in control conditions (21.99‐fold increase). No significant differences were observed in GGPPS gene expression in the WT line, which remained low in all tested conditions.

Figure 7.

Transcriptional expression levels of key genes involved in the biosynthetic pathway of paclitaxel and related taxanes of three different T. baccata cell lines in elicited (_E) and control conditions (_C): WT, wild type line; DBTNBT, a 3′‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing line; and BAPT, a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing line. Cell cultures were maintained for 24 days and elicitation was performed with 1 μm coronatine +50 mm β‐methyl‐cyclodextrins. The relative gene expression levels were normalized with respect to the same cell line growing for 12 days in the growth medium (GM) without elicitors. Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey's honestly significant difference test. The gene names are as follow: GGPPS (a), geranylgeranyl diphosphate synthase; TXS (b), taxadiene synthase; DBAT (c), 10‐deacetylbaccatin III 10‐O‐acetyltransferase; T7βOH (d), taxane 7‐β‐hydroxylase; BAPT (e), baccatin III‐3‐amino‐13‐phenylpropanoyltransferase; DBTNBT (f), 3′‐N‐debenzoyltaxol N‐benzoyltransferase; and PAM (g), phenylalanine aminomutase.

The transcript levels of the TXS gene in the elicited DBTNBT line increased steadily until a peak of expression at 48 h (51.15‐fold increase), remaining constant thereafter (Figure 7b). In the elicited BAPT cell line, TXS mRNA levels stayed relatively low until 48 h (55.33‐fold increase), after which they sharply decreased. Similarly, low transcript levels were observed in the elicited WT cell cultures until 48 h (21.32‐fold increase), but unlike the BAPT line, they remained high thereafter.

The expression pattern of the DBAT gene (Figure 7c) in elicited transgenic lines was similar to those of the previous genes. The highest expression was found in the elicited DBTNBT cell cultures, reaching a maximum at 72 h (52.79‐fold increase) after a steady increase from the beginning of the experiment. Unelicited DBTNBT cell cultures exhibited low DBAT gene expression rates until 48 h, when they increased, peaking at 72 h (19.23‐fold increase). In the BAPT line, although the DBAT gene expression was not clearly enhanced by elicitation, two peaks were detected, one at 12 h (8.18‐fold increase) and the highest at 48 h (12.30‐fold increase); in control conditions, only one peak was detected, at 6 h (8.32‐fold increase). In elicited WT cell cultures, the only significant peak of gene expression occurred (8.15‐fold) at 12 h post‐elicitation.

The mRNA levels of the T7βOH gene remained consistently high from 6 to 12 h (100‐fold higher) in elicited DBTNBT cell cultures, decreasing by half at 24 h (50.21‐fold higher) before increasing again to reach the highest values at the end of the study (299‐fold and 445‐fold at 48 and 72 h, respectively) (Figure 7d). In the control DBTNBT line, this hydroxylase was only highly expressed at 48 h (28.97‐fold increase) and 72 h (70.73‐fold increase), indicating a later and lower genetic response in the absence of elicitors. Interestingly, the second highest expression level was detected in the WT line. In elicited WT cell cultures, two significant peaks occurred, at 12 h (213.78‐fold increase) and 48 h (154.97‐fold increase), whereas in control conditions one peak was observed at 48 h (191.34‐fold). Conversely, the T7βOH gene was least expressed in the BAPT line, with relatively small increases observed at 12 h (62.25‐fold) and 48 h (80‐45‐fold) after elicitation.

The flux‐limiting gene BAPT was very strongly expressed in the transgenic BAPT line throughout the study, reaching a maximum at 12 h (53.24‐fold) and 48 h (59.72‐fold) under elicitation. Considering the low levels of BAPT gene expression in natural conditions (non‐elicited and non‐overexpressed), these results confirm a high BAPT gene overexpression under the control of a CaMV 35S promoter (Figure 7e). The BAPT transcript levels in the control BAPT line (average 18‐fold increase) were enhanced by elicitation: 8.84‐fold at 12 h and 2.36‐fold at 48 h. In the other lines, the BAPT gene remained poorly expressed throughout. In the elicited DBTNBT line, BAPT gene expression increased steadily but remained low at 72 h (15.18‐fold increase), with lower expression observed in control WT and DBTNBT lines (about 4‐fold increase).

Similarly, constitutive overexpression of the DBTNBT gene in the control DBTNBT cell line (16‐fold increase on average) was higher than in natural conditions (Figure 7f), and was significantly enhanced by elicitation at 48 h (49.72‐fold increase) and 72 h (61.93‐fold increase), which represents a 3.10‐fold and 3.87‐fold increase, respectively, with respect to the control. In the other lines, only the elicited BAPT line showed a significant peak of gene expression, at 48 h (12.21‐fold increase).

Finally, it is noteworthy that PAM gene expression was only significantly higher in the elicited BAPT line from 6 to 48 h, reaching maximum values at 12 h (81.77‐fold increase) and 48 h (48.30‐fold increase) (Figure 7g). In the control BAPT line, PAM transcript levels were only significantly enhanced at 6 h (25.44‐fold). Interestingly, in this line, the time points of maximum activity of the PAM gene coincided with those of peak BAPT gene expression under the same culture conditions. In contrast, none of the other lines showed an increase in PAM gene expression higher than 10‐fold at any experimental time point.

Discussion

To the best of our knowledge, this is the first report of a successful transformation of T. baccata seedlings by direct inoculation using two modified R. rhizogenes A4 strains harbouring taxane genes in their plasmids. According to the literature, only He et al. (2023) have previously managed to obtain T. baccata hairy roots using R. rhizogenes A4, although in that case the wild type strain was used, and the infection process was performed in ex vitro conditions.

In the present study, a total of eight root lines appeared after 9–11 months of bacterial inoculation, resulting in a root induction frequency of 10%. Only 25% of the roots were confirmed as transformed by PCR analysis and only one was stable enough to be maintained as a root culture, which represents a success rate of 2.5% in the whole infection process. This low efficiency of direct infection matches previous reports on Taxus transformation rates. He et al. (2023) also achieved a low transformation efficiency of 14.3% in ex vitro T. baccata seedlings. Furmanowa and Syklowska‐Baranek (2000) obtained a 3% transformation frequency of T. × media var. Hicksii seedlings after 19 weeks of direct inoculation with the R. rhizogenes strain LBA 9402. Similarly, Kim et al. (2009) reported that the direct infection method generated the highest rate of hairy roots (approximately 26%), although most of them were unviable in the successive cultures.

The low success rates of these studies can be explained by the difficulty of transforming gymnosperms and the slow growth of their roots, both adventitious and hairy (Zavattieri et al., 2016). Magnussen et al. (1994) observed that hairy or highly branched roots were formed only in 2%–3% of Picea abies, Pinus sylvestris and P. contorta seedlings inoculated with R. rhizogenes R1600. In another study, Mihaljevic et al. (1999) improved the rooting process of Sequoia sempervirens by infecting basal cut ends of 2‐cm‐long shoot tips with R. rhizogenes 8196 manopine strain. After 2–4 months, roots were formed on 58%–69% of the shoots, but none had the hairy root phenotype.

Although T. baccata hairy root lines were obtained, they were unsuitable for a biotechnological production system due to their slow growth rate and low taxane yield. The roots were therefore dedifferentiated by applying plant growth regulators and the resulting calli were used to establish cell suspension cultures. Exposito et al. (2010) successfully obtained transformed hairy root cultures of T. x media overexpressing the T. baccata TXS gene, but were forced to dedifferentiate them into callus lines due to their slow growth rate. Also, Chang et al. (2017), despite obtaining T. sumatrana hairy root lines, observed a higher production of DABIII, BACIII and PTX in calli.

Cell suspension cultures are one of the most effective and adaptable methods for the production of paclitaxel and related taxanes in Taxus spp. However, the yields are limited by pathway bottlenecks (Kashani et al., 2018). These authors identified DBAT, BAPT and DBTNBT as the genes encoding the most important rate‐limiting enzymes in T. baccata cell suspension cultures elicited with COR and randomly methylated‐β‐CDs. Supporting their results, in the present study the overexpression of DBTNBT and BAPT genes in long‐term and stable T. baccata cell cultures clearly increased the total taxane content at days 8 and 16 of culture in unelicited conditions. However, despite the improvement induced by the overexpression of the target genes, yields could be enhanced further by elicitation, as previously reported in T. x media cell cultures transformed with the TXS gene (Exposito et al., 2010) or T. mairei cells transformed with TXS and DBAT genes (Ho et al., 2005). Accordingly, the highest taxane yield of 367 mg/L was obtained in the elicited DBTNBT‐overexpressing line, 308.15 mg/L of which corresponded to paclitaxel, at day 8 of culture, which was earlier than in the other cell lines. In the elicited BAPT line, the total taxane production reached a peak of 181.56 mg/L at day 16, including 134.01 mg/L of paclitaxel. In this sense, the PTX values reported in this study greatly exceed those obtained by previous authors. For example, Ketchum et al. (1999) achieved 117 mg/L of PTX in elicited T. canadensis cell cultures with 200 μm MeJA, which is 2.63‐fold and 1.15‐fold times lower than the amount produced by DBTNBT‐ and BAPT‐overexpressing lines, respectively. Similarly, Sabater‐Jara et al. (2014) and Escrich et al. (2018) detected 65 mg/L and 90 mg/L of PTX in T. media cell cultures under combined elicitation experiments with 100 μm MeJA +50 mm β‐CDs and 1 μm COR + 10 mg/L calix[8]arenes, respectively, which are still 4.74‐fold and 3.42‐fold times lower than the maximum PTX content produced by DBTNBT‐overexpressing line.

Based on these results, it can be deduced that the taxane pathway bottleneck was overcome more effectively by overexpressing the DBTNBT gene than the BAPT gene, as it led to a higher levels of paclitaxel and total taxanes, with an earlier peak of production. It can be speculated that DBTNBT overexpression seems to reduce the feedback repression of other genes, thus resulting in the production of more paclitaxel (Cusido et al., 2014). On the other hand, it was verified that the overexpression of one limiting gene of the pathway does not stimulate the expression of the other, as the transcript levels of the BAPT gene remained low in the DBTNBT‐overexpressing line even under elicitation, and vice versa in the BAPT line. It has been shown that this low gene expression has an epigenetic origin due to methylation processes in the Y‐Patch promoter region of both genes (Escrich et al., 2022; Sanchez‐Munoz et al., 2018). However, the high constitutive gene expression observed in the transformed lines, both control and elicited, could suggest this methylation was absent in the genes obtained through bacterial transformation.

Nevertheless, the increase in paclitaxel production in both transgenic lines may not only be due to the overexpression of genes located downstream in the biosynthetic pathway, but also genes involved in early and intermediate steps, as can be inferred from the transcript profile analysis. The GGPPS gene was highly overexpressed in both elicited lines and its transcripts showed long half‐lives, which could result in an overproduction of all diterpenoids, including substrates required for paclitaxel biosynthesis. In agreement with other authors, the high fold change observed in TXS gene expression, especially in the DBTNBT line, may suggest that this gene does not encode a rate‐limiting enzyme (Exposito et al., 2010; Kashani et al., 2018). However, the overproduction of taxadiene and other intermediates of paclitaxel may stimulate an increased synthesis of related hydrolytic enzymes to prevent their excessive accumulation (Kashani et al., 2018). Accordingly, the steady and gradual increase in DBAT and T7βOH gene expression observed in the DBTNBT line may explain why intermediate substrates, such as DABIII and BACIII, did not accumulate in a significant level in the cell cultures, suggesting their rapid conversion and consumption in downstream reactions. In contrast, the relative expression of most of the genes analysed in the BAPT line showed a pattern of isolated peaks of expression, generally at 12 and 48 h after elicitation. Therefore, the relatively low half‐life of the transcripts may be one of the reasons why the paclitaxel levels achieved in this line were lower. Interestingly, a correlation was detected between the maximum activity of BAPT and PAM genes in the BAPT line. As the expression of the PAM gene in the DBTNBT line remained consistently low, a positive effect of the BAPT gene overexpression on the activity of this gene can be inferred, given that increased formation of complex taxanes such as paclitaxel, CEPH or DAT requires a higher rate of side chain synthesis.

Finally, another key factor involved in the enhancement of paclitaxel production was the use of a dual elicitation treatment. Thus, the action of COR, a potent elicitor able to trigger the jasmonate signalling pathway (Zhao et al., 2003), was combined with that of CDs, a family of cyclic oligosaccharides that can form inclusion complexes with hydrophobic compounds such as paclitaxel and thus facilitate their excretion from the cells to the culture medium (Ramirez‐Estrada et al., 2015). The high levels of extracellular paclitaxel found inside these complexes (ranging from 60% to 90%) in the elicited samples not only reduced its cellular toxicity and the feedback repression on upstream genes such as GGPPS, TXS and T7βOH, but also prevented its degradation in the culture medium (Gajula et al., 2018).

Material and methods

Plant material

Mature seeds of Taxus baccata (Figure 1a), were surface‐sterilized as described by Cusido et al. (2002). For germination, the intact embryos were extracted from the sterilized seeds, mounted on hormone‐free modified DCR medium (Gupta and Durzan, 1985; Syklowska‐Baranek et al., 2009), solidified with (5 g/L) Phyto agar (Duchefa Biochemie, Haarlem, The Netherlands), and maintained in darkness at 25 °C. Germination occurred after 2–3 weeks and once the seedlings reached the Petri dish cap, they were transferred into Magenta™ vessels (Sigma Aldrich, St Louis, MO, USA) containing the same medium and kept at 25 °C with a 16 h/8 h light/dark photoperiod.

Bacteria strains and plasmids

The plant material was infected with two different A4 R. rhizogenes engineered strains harbouring, together with the plasmid pRiA4, the binary expression vectors pK7WG2 and pCAMBIA_1301 for the BAPT and DBTNBT genes, respectively. For DBTNBT gene overexpression, the full coding sequence of the DBTNBT gene from T. media (AY563629.1; 1316 bp) was introduced within the backbone of the pCAMBIA_1301 plant expression vector (AF234297; 11 849 bp) (Figure S11). For BAPT gene overexpression, the full coding sequence of the BAPT gene from T. cuspidata (KC988329.1; 1337 bp) was introduced within the backbone of the pK7WG2 plant expression vector (11 191 bp) obtained through the GATEWAY™ conversion technology (Karimi et al., 2002) (Figure S12). The expression of both genes was controlled by one copy each of the constitutive Cauliflower mosaic virus 35S promoter (CaMV 35S). For the selection of transformed bacteria and plants, the plasmids contained the following antibiotics: spectinomycin for bacteria and kanamycin (nptII) for plants (pK7WG2‐BAPT), as well as kanamycin for bacteria and hygromycin B (hptII) for plants (pCAMBIA_1301‐DBTNBT).

Stable transformation of Taxus plantlets and establishing hairy root cultures

The transformed R. rhizogenes strains were grown for 72 h at 28 °C in solid YEB medium (van Larebeke et al., 1977) containing (100 mg/L) kanamycin (pCAMBIA_1301‐DBTNBT) or (75 mg/L) spectinomycin (pK7WG2‐BAPT). Bacterial YEB medium consisted of (5 g/L) beef extract, (1 g/L) yeast extract, (5 g/L) peptone, (5 g/L) sucrose and 2 mm MgSO₄.The pH was adjusted to 6.8 prior to autoclaving. All reagents used for medium preparation were from Becton, Dickinson and Company (Sparks, MD).

The shoot tips, young leaves and hypocotyls of 8‐week‐old Taxus seedlings were wounded with a sterile needle containing the bacterial solution (Figure 1b), as described by Furmanowa and Syklowska‐Baranek (2000). After infection, Taxus seedlings were incubated for 48 h in solid hormone‐free modified DCR medium (Gupta and Durzan, 1985; Syklowska‐Baranek et al., 2009), then transferred to the same medium supplemented with (500 mg/L) cefotaxime to eliminate the agrobacteria, and maintained in a 16 h/8 h light/dark photoperiod at 25 °C. Putative transformed hairy roots emerged from the wounded sites 9–11 months after infection (Figure 1c) and when 2–3 cm long they were excised and cultured individually in the same medium supplemented with (500 mg/L) cefotaxime (Figure 1d). Taxus roots were subcultured every 2 weeks and after 4 months the antibiotic was removed.

Induction and establishment of callus cell lines

Due to the slow growth rate of the T. baccata roots, 5‐7‐cm‐long sections of established root lines (Figure 1e) were excised and placed in contact with callus induction medium, which consisted of Gamborg's B5 medium (Gamborg et al., 1968) supplemented with 2× B5 vitamins, 3% sucrose and the growth regulators (4 mg/L) 2,4‐dichlorophenoxyacetic acid, (1 mg/L) kinetin and (0.5 mg/L) gibberellic acid, as described by Exposito et al. (2010). After 3–4 weeks, callus tissues (Figure 1f,g) were separated from the explants and placed together (Figure 1h). Calli were grown in solid Gamborg's B5 growth medium (GM) (Gamborg et al., 1968) supplemented with 2× B5 vitamins, 0.5% sucrose, 0.5% fructose, and the growth regulators: (2 mg/L) 1‐naphthaleneacetic acid, (0.1 mg/L) 6‐benzylaminopurine and (0.5 mg/L) gibberellic acid (Exposito et al., 2010).The cells were cultured in the GM at 25 °C in darkness and subcultured every 2 weeks to obtain enough friable and vigorous calli to establish cell suspension cultures.

To reduce excessive production and release of polyphenols into the medium, an antioxidant solution (Kim et al., 2005) consisting of L‐glutamine (14.6 g/L), ascorbic acid (2.5 g/L) and citric acid (2.5 g/L) was added to both the induction medium and GM after autoclaving. The solution was previously filter‐sterilized (0.22 μm sterile PES filters; Millipore, Billerica, MA).

Molecular confirmation of transgenic lines by PCR

Genetic transformation of all Taxus root and callus lines obtained were confirmed by PCR analysis using the DreamTaq Green PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) with 0.5 μg DNA. Previously, genomic DNA was extracted from samples as in Dellaporta et al. (1983). Specific primers were used for the amplification of the bacterial genes (rolA, rolB, rolC, aux1 and virD), antibiotic resistance genes (nptII and hptII) and the endogenous taxane gene BAPT. Additionally, for further verification of the transformation, a specific region was amplified comprising CaMV 35S and the two taxane genes within the bacterial plasmids: 35S‐DBTNBT and 35S‐BAPT (Table S1). For the DBTNBT gene, it should be noted that only the 35S‐DBTNBT primer pair could be used because no other coding sequence was available in the NCBI database (GenBank: AY563629.1). The conditions of the amplification reaction were the following: denaturation at 95 °C for 5 min, 40 cycles of 95 °C for 1 min, Tm (specific for each primer pair; see Table S1) for 1 min and 72 °C for 1 min, with a final step at 72 °C for 5 min. PCR products were analysed by electrophoresis on 1% agarose gels.

Root cultures and elicitation treatments

Root culture experiments were carried out as in Syklowska‐Baranek et al. (2022) with slight variations. Briefly, transgenic L9 and wild type (untransformed) L4 roots were cultivated on a sterile rounded stainless steel grid in 250 mL Erlenmeyer flasks containing 30 mL of hormone‐free modified liquid DCR medium (Gupta and Durzan, 1985; Syklowska‐Baranek et al., 2009) and routinely subcultured every 4 weeks. For the elicitation experiments, inocula of 0.5 ± 0.05 g FW of 4‐week‐old roots were transferred to 30 mL of fresh DCR medium for 28 days. 1 week before the addition of elicitors, roots were primed with 100 μm β‐aminobutyric acid (BABA). Elicitation was performed at day 28 of culture with a combination of 1 μm coronatine (COR) and 50 mm of randomly methylated‐β‐cyclodextrins (β‐CDs), supplemented with the precursors 100 μm L‐phenylalanine and 10 μm sodium nitroprusside. Control cultures (without elicitation) were also treated with BABA and precursor feeding. All reagents were obtained from Sigma Aldrich (St Louis, MO). Due to the small amount of biomass available, samples were collected only at day 0 and at day 14 of elicitation for taxane analysis, the time point of maximum paclitaxel yield achieved in the aforementioned studies.

Cell suspension culture conditions and elicitation treatments

Transgenic and non‐transgenic (control) cell lines were cultured using a two‐stage system as described by Cusido et al. (2002) and Palazon et al. (2003). Briefly, after 14 days of cultivating the T. baccata cells in liquid GM for biomass production, 3 g of cells were transferred into 10 mL of taxane production medium (PM) in a 175 mL flask capped with a Magenta™ B‐cap (Sigma Aldrich). The PM consisted of Gamborg's B5 liquid medium (Gamborg et al., 1968) supplemented with 3% sucrose and the growth regulators (2 mg/L) picloram, (0.1 mg/L) kinetin and (0.5 mg/L) gibberellic acid at pH 5.8. The antioxidant solution was also added following the same proportion already described. Cell cultures were kept in the dark at 25 ± 0.2 °C and 100 ± 1 rpm in an orbital shaker‐incubator (Adolf Kühner, Sch weiz).

Taxane production was stimulated by adding the elicitors at the beginning of the second phase of culture (Onrubia et al., 2013). As with root cultures, elicitation was also performed with 1 μm COR and 50 mm of β‐CDs (Sigma Aldrich), which were filter‐sterilized before addition to the cell suspensions. Samples were harvested at days 0, 8, 12, 16, 20 and 24 of culture.

Biomass determination and viability assay

Fresh weight (FW) was determined by filtering the cells with 80 μm Nylon filters. The cells were freeze‐dried to obtain the dry weight (DW) and perform the taxane extraction. Cell viability was evaluated as described by Exposito et al. (2010) and was determined at the beginning (day 8), middle (day 16) and end (day 24) of the experiments.

The hairy root growth was determined based on the index described by Syklowska‐Baranek et al. (2022). In control conditions, before elicitation, growth was determined according to an increase in FW (FWI) as following:

$$\mathrm{FWI}={\mathrm{FW}}_{28\mathrm{d}}/{\mathrm{FW}}_{\text{0d}}$$

where FW_28d is the FW of hairy roots at day 28 of culture before elicitation and FW_0d is the FW on the day of inoculation. The variation of FW under elicitation (FWE) was evaluated as following:

$$\mathrm{FWE}={\mathrm{FW}}_{14\mathrm{d}}/{\mathrm{FW}}_{\text{0d}}$$

where FW_14d is the FW of hairy roots at day 14 of elicitation and FW_0d is the FW on the day of inoculation (42 days before).

Total taxane extraction and quantification

Taxanes were extracted from the culture media and lyophilized biomass as in Perez‐Matas et al. (2022). The same protocol was followed for hairy roots but adjusting the volumes of reagents to 30 mL of culture medium and the total quantity of lyophilized roots (from 50 to 80 mg DW). Finally, all samples were resuspended in 500 μL of MeOH and filtered (0.45 μm PVDF filters; Millipore) prior to analysis. HPLC analyses were performed as in Sabater‐Jara et al. (2014). Taxanes were quantified by integrating the corresponding peak of each target compound in the standard calibration curve: 10‐deacetylbaccatin III (DABIII), baccatin III (BACIII), 10‐deacetyltaxol (DAT), cephalomannine (CEPH) and paclitaxel (PTX). All standards were obtained from Abcam (Cambridge, UK).

Transcript analysis

Total RNA was isolated from 200 mg of frozen cells at 0, 6, 12, 24, 48 and 72 h using the Real Plant RNA Kit (REAL, Valencia, España) according to the manufacturer's instructions. The RNA concentration of each sample was determined using a NanoDrop ND‐1000 spectrophotometer (NanoDrop Technologies Wilmington, DE). cDNA was prepared from 1 μg of RNA with SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, MA, USA).

The expression level of key taxane biosynthetic genes (GGPPS, TXS, DBAT, BAPT, DBTNBT, T7βOH and PAM genes) was determined by qRT‐PCR using SYBR Green Mastermix (Biorad, Hercules, CA) in a 384‐well platform system (LightCycler® 480 Instrument; Roche, Basel, Switzerland). Gene‐specific primer sequences were obtained from previous studies of our research group (Onrubia et al., 2010, 2011; Sabater‐Jara et al., 2014; Table S2). The reaction mixture, primer amplification efficiency and thermos‐cycling program were as described previously (Perez‐Matas et al., 2022). The TBC41 gene was selected as a reference to normalize gene expression (Sabater‐Jara et al., 2014; Vidal‐Limon et al., 2018; Yanfang et al., 2018). Data were analysed using LightCycler® analysis software v4.1. Expression levels are presented as fold change values relative to the baseline of non‐elicited conditions (day 12 of culture in the optimal growth medium).

Statistics

All data concerning biomass determination, cell viability, taxane quantification and transcription profiling were expressed as the average of three independent determinations ± standard deviation (n = 3 ± SD). Statistical analysis was carried out using Excel and RStudio software. Multifactorial ANOVA followed by Tukey's post hoc multiple comparison tests were used for statistical comparisons. A P‐value of <0.05 was assumed for significant differences.

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

EP‐M performed the experiments and contributed to the design of the experimental work. JP, MB and DH‐M conceived and designed the experimental work and wrote the manuscript. MB and EM supervised the work, read and commented the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This research was funded by Agencia Estatal de Investigación. REF: PID2020‐113438RBI00/AEI/10.13039/501100011033 and by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) del Departament de Recerca i Universitats de la Generalitat de Catalunya 2021 SGR00693. D.H.‐M. is a Postdoctoral researcher at María Zambrano at the University of Barcelona. His contract is financed by the Ministry of Universities, the European Union Next GenerationEU/PRTR.i and the Recovery, Transformation and Resilience Plan.

Supporting information

Figure S1 Molecular verification of the transgenic nature of the hairy root and callus lines.

Figure S2 Morphology of Taxus baccata transgenic (A) and untransformed (B) hairy root lines after 1 year of culture.

Figure S3 Molecular confirmation of overxpression of DBTNBT and BAPT genes in Taxus baccata lines L9 and L6.

Figure S4 Molecular verification of the genetic integration of the T‐DNA of the plasmids pCAMBIA_1301 and pK7WG2 in L9 and L6 roots and calli by PCR amplification of the antibiotic resistance genes hptII (a) and nptII (b).

Figure S5 Determination of fresh weight (FW) indices FWI and FWE of two Taxus baccata root lines (WT/DBTNBT) in the initial 28 days of growth in DCR medium and after 14 days of elicitation, in elicited (_E) and control conditions (_C): WT, wild type root line; and DBTNBT, a 3'‐N‐debenzoyltaxol N‐benzoyltransferase‐overexpressing hairy root line.

Figure S6 Contents of the different taxanes studied (DABIII, BACIII, DAT, CEPH and PTX) expressed as total contents (a), total taxanes found in the liquid medium (b), or total taxanes accumulated in the cells (c) throughout the experiment in two different Taxus baccata root lines in elicited (_E) and control conditions (_C): WT, wild type hairy root line; and DBTNBT, a 3'‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing hairy root line.

Figure S7 Taxane excretion rates expressed as percentage of total taxanes found inside the cells (blue colour) and in the liquid medium (orange colour) of three different T. baccata cell lines in elicited (_E) and control conditions (_C): WT, wild type line; DBTNBT (DBT.), a 3'‐N‐debenzoyltaxol N‐benzoyltransferase overexpressor line; and BAPT, a baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene overexpressor line.

Figure S8 Contents of the different taxanes studied (DABIII, BACIII, DAT, CEPH and PTX) expressed as total taxanes found in the liquid medium (a) or total taxanes accumulated in the cells (b) throughout the experiment in the wild type T. baccata cell line (WT) in elicited (WT_E) and control conditions (WT_C).

Figure S9 Contents of the different taxanes studied (DABIII, BACIII, DAT, CEPH and PTX) expressed as total taxanes found in the liquid medium (a) or total taxanes accumulated in the cells (b) throughout the experiment in the T. baccata 3'‐N‐debenzoyltaxol N‐benzoyltransferase gene‐overexpressing cell line (DBTNBT) in elicited (DBTNBT_E) and control conditions (DBTNBT_C).

Figure S10 Contents of the different taxanes studied (DABIII, BACIII, DAT, CEPH and PTX) expressed as total taxanes found in the liquid medium (a) or total taxanes accumulated in the cells (b) throughout the experiment in the T. baccata baccatin‐aminophenylpropanoyl‐13‐O‐transferase gene‐overexpressing cell line (BAPT) in elicited (BAPT_E) and control conditions (BAPT_C).

Figure S11 pCAMBIA_1301 vector map showing the enzyme restriction sites, kanamycin resistance gene for bacterial selection, hygromycin B resistance gene for plant selection and the gusA reporter gene.

Figure S12 pK7WG2 vector map showing the enzyme restriction sites, spectinomycin resistance gene for bacterial selection and kanamycin resistance gene for plant selection (GATEWAY^TM technology; Karimi et al., 2002).

Table S1 Sequences of the primers used for PCR amplification of target genes.

Table S2 Sequences of the primers used to amplify the genes by RT‐qPCR.

Data availability statement

The raw data supporting the results of this article will be made available by the authors, without undue reservation.