Enhancement of taxane production in cell suspension culture of Corylus avellana L. Kalınkara by elicitation and precursor feeding

Abstract Paclitaxel (trademark Taxol®) is the most effective chemotherapeutic agent against a wide range of cancers. Hazelnut (Corylus avellana L.) cell culture is a promising and inexpensive strategy for producing paclitaxel and related taxanes. This study evaluated the effect of elicitation and taxane precursor feeding for the enhancement of taxanes in a Turkish hazelnut (C. avellana cv. ‘Kalınkara’) cell suspension culture. Elicitor methyl jasmonate (100, 200 and 300 µmol L−1) and precursor phenylalanine (3μmol, 3 and 6 mmol L−1) were tested in cell suspension cultures. While methyl jasmonate increased the accumulation of taxanes, especially cephalomannine, via induction of the paclitaxel biosynthetic pathway, phenylalanine slightly induced the conversion of 10-deacetylbaccatin III and baccatin III to the end product paclitaxel (0.5349 µg g−1). The results showed that 200 µmol L−1 methyl jasmonate led to significantly greater induction of taxanes than 100 and 300 µmol L−1. The amount of cephalomannine (331.6 µg g−1) in cell suspension cultures elicited by 200 µmol L−1 methyl jasmonate was 8-fold higher than that in the control (41.61 µg g−1), while 10-deacetylbaccatin III (6.174 µg g−1) and baccatin III (3.956 µg g−1) increased almost twice as much as the control. The highest total paclitaxel yield was 5.453 µg g−1 in the same culture conditions. In conclusion, cell culture systems of C. avellana L. cv. ‘Kalınkara’ hazelnut, established for the first time, may become a potential candidate for industrial-level production of cephalomannine and other taxanes with further optimization.


Introduction
Paclitaxel (PTX), also known by the commercial name Taxol ® , is the best-selling anti-cancer drug clinically applied for the treatment of various types of cancers and seems promising in treating non-cancer diseases, such as skin disorders, renal and hepatic fibrosis, inflammation, axon regeneration, limb salvage, and coronary artery restenosis [1,2].PTX disrupts the normal microtubule dynamics during cell division and the interphase by promoting tubulin polymerization and causing cell cycle arrest and apoptosis [3,4].PTX is a complex diterpene alkaloid first extracted from Taxus brevifolia bark in 1967 [5].However, Taxus (yew) trees grow slowly, and harvesting the bark is destructive; therefore, commercial production of PTX by harvesting yew bark has threatened Taxus species with extinction.Eight mature yew trees are needed to obtain enough Taxol ® throughout natural extraction to treat only one patient [6,7].Moreover, chemical synthesis is extremely challenging because of its complex nature [8].PTX biosynthesis involves 19 metabolic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate (GGPP).GGPP is cyclized by taxadiene synthase (TXS) to form the first intermediate taxadiene.Following the formation of the taxane skeleton, several metabolic steps, including hydroxylation, acyl/ aroylation, epoxidation, and oxidation, yield the functionally important intermediates 10-deacetyl baccatin III (10DAB) and baccatin III (BACIII).Then, the attachment of a side chain derived from β-phenylalanine at the C13-OH of BACIII generates the chemotherapeutic agent PTX and a new drug candidate, cephalomannine (CEPH).To date, most steps of the taxane biosynthetic pathway have been elucidated, but further in-depth studies are still required to confirm the suitability of uncharacterized candidate genes [9][10][11][12].
Plant cell suspension cultures offer an efficient and sustainable approach for the mass production of taxanes [13][14][15].Although the semi-synthesis of Taxol ® from its precursors, such as 10DAB, BACIII and CEPH, is currently used efficiently, it is a very expensive and time-consuming process [16].Although Taxol ® production through Taxus cell culture is performed biotechnologically at the industrial level by Phyton Biotech, ESCAgenetic, Samyang and Nattermann, there is still a high demand for this drug [17][18][19][20].A natural congener of PTX, namely CEPH [21,22], also has anti-cancer properties.Owing to structural similarities, CEPH shows anti-tumor activity comparable to that of PTX in several different cell lines [23].The cytotoxic effects of CEPH were first demonstrated in human glioblastoma cells [24].Zhang et al. [25] have demonstrated that CEPH significantly attenuates hepatocellular carcinoma progression in vitro and in vivo.In addition, CEPH has been shown to inhibit proliferation and invasion in vitro and bone metastasis in prostate cancer in vivo [26].Therefore, CEPH has recently been evaluated as an anti-cancer drug candidate [25][26][27][28][29].
The search for new taxane sources is considered crucial to meet rapidly rising worldwide demand.In addition to Taxus species, hazelnut (Corylus avellana) has also been described as another plant source among angiosperms that produces PTX, CEPH and related taxanes [30,31].Seven Turkish hazelnut cultivars have been shown to contain PTX, CEPH and other taxane derivatives in different tissues [32].Although the concentration of taxanes in hazelnuts is low compared to the bark of Taxus spp., there are major advantages to producing taxanes by hazelnut cell cultures.Hazelnut trees are widely available, fast-growing in vivo, and more easily cultivated in vitro than yew trees [33][34][35].In vitro culture of C. avellana can be a promising and inexpensive method for PTX production [36][37][38].Among various biotechnological advances developed for taxane yield enhancement in in vitro plant cultures [39][40][41], the most efficient strategy is elicitation carried out with biotic and abiotic elicitors [42][43][44].Biotic elicitors, such as jasmonates, are widely applied for eliciting the biosynthesis of secondary metabolites in in vitro culture, owing to their high efficacy and low toxicity to plant cells [45][46][47].Jasmonates, particularly MeJA, are defined as plant hormones because they induce cellular responses at low concentrations distant from their site of synthesis and are also widely known to elicit a wide range of compounds by inducing the expression of plant genes for various biosynthetic pathways [42].On the other hand, precursor feeding is another strategy for increasing taxane production.Phenylalanine (Phe) is a precursor of the PTX biosynthetic pathway and is involved in the production of PTX side chains [9].The enhancement of taxoid accumulation through precursor feeding in Taxus cell cultures has opened the possibility of inducing the same pathway in hazelnuts [48].Increased PTX production was observed in C. avellana cell cultures with Phe and MeJA [36,37,49].Enhancing the biosynthesis of these metabolites by precursor feeding or stimulating the expression of genes associated with the pathway is an obvious way forward, but is not always possible because of the limited amount of information available.Although MeJA and Phe have been tested in C. avellana cultivars previously, they have not been tested in Turkish hazelnut cultivars, which are notable for their taxane production [32].In the present study, cell suspension cultures of a taxane-containing Turkish hazelnut cultivar, Kalınkara, were established for the first time.The potential of elicitation and precursor feeding to increase valuable taxanes was investigated.The accumulation of taxanes (10DAB, BACIII, PTX and CEPH) was analyzed by high-performance liquid chromatography (HPLC), and the impact of the elicitor MeJA and precursor Phe on taxane accumulation was discussed.

Plant material
Corylus avellana cv.'Kalınkara' hazelnuts were kindly provided by Prof. Dr. Umit SERDAR from Ordu province (40°59′27.9′′N37°35′01.6′′E),Turkey in 2018.The seeds were separated from their green husks.Dry and mature seeds with a mean diameter of 17 mm were used for in vitro cultivation.They were stored in a fridge at 4 °C until experimentation.

Callus cultures
The mature seeds with hard-brown shells were washed in tap water for 1 h.After that, they were taken into a sterile laminar flow cabinet.The seeds were separated from their hard-brown shells, and they were surface-sterilized with 70% ethanol (v/v) for 30 s, submerged in 3% (v/v) NaOCl, including a few drops of Tween20, for 30 min, and rinsed three times with sterile distilled water.Then, the seeds were sterilized with ethanol 99% (v/v) for a few seconds and rinsed again three times with sterile distilled water.The endosperms of sterilized seeds were trimmed and cut into four pieces in a Petri dish with sterile filter paper.The cut surfaces of explants were placed onto MS medium [50] supplemented with 30 g L −1 sucrose, 2 mg L −1 2,4-dichlorophenoxy acetic acid (2,4-D, Sigma, D7299) and 0.2 mg L −1 6-benzylamino purine (BAP, Sigma, B3274) solidified with 8 g L −1 agar (Sigma, A1296).The pH of the medium was adjusted to 5.7 ± 0.1 with either NaOH or HCl before autoclaving for 15 min at 121 °C.BAP and 2,4-D were sterilized by filtering through 0.22 µm filters (Millipore, GSWP04700).Callus cultures were kept at 25 ± 1 °C in a growth chamber (70% humidity, 25 ± 1 °C, 16 h light/8 h dark, 1400 lx, Sanyo, MLR-352H), and sub-cultured routinely at four-week intervals.

Cell suspension cultures
The cell suspension cultures were initiated by transferring white and friable 5 g callus into 250 mL Erlenmeyer flasks containing 50 mL MS medium supplemented with 2 mg L −1 2,4-D and 0.2 mg L −1 BAP.The flasks were incubated at 25 ± 2 °C on an orbital shaker (Labnet, 211DS), at 120 rpm at 25 °C, and they were routinely sub-cultured every 30 days until the cells reached homogeneity.
The growth patterns of cultures were characterized by the cumulative biomass increment and were modeled by a sigmoidal curve.Every five days, 10 mL cell suspension cultures were transferred to 15 mL Falcon tubes.They were centrifuged for 15 min at 9500 rpm (Beckman Coulter, Allegra X-22R), supernatants were discarded, and then the tubes were incubated for 48 h at 65 °C for dry weight measurements.

Elicitation
On the 17th day of cultivation, MeJA (Sigma, 392707) and Phe (Sigma, P1751) were separately added to the flasks containing 50 mL of cell suspension cultures to give final concentrations of 100, 200 and 300 µmol L −1 of MeJA and 3 µmol L −1 , 3 mmol L −1 and 6 mmol L −1 of Phe.The same volumes of distilled water were added to cultures that were used as a control.MeJA and Phe were dissolved in distilled water and then sterilized by filtering through 0.22 µm filters (Millipore, GSWP04700).The cultures were harvested on the 30th day of cultivation for HPLC analysis.

Extraction and analysis of taxanes
Taxane extraction was performed as described by Gallego et al. [36].All of the cell suspension culture samples were frozen at −80 °C for extraction of taxanes.The samples were crystallized at −56 °C for 48 h using a lyophilisator (Christ, 1-2LDPlus).A total of 50 mg of freeze-dried biomass was extracted with 1 mL of 90% methanol/water (v/v, Sigma, 34885).The cells were disrupted using a mixer mill (Domel Tehtnica, MillMix 20) for 2 min at 1800 rpm.After centrifugation (10 min at 12.000 rpm), the methanol extracts were transferred to a new Falcon tube.The pellets were re-extracted again as described above, and the supernatants were collected in the same Falcon tubes.Afterward, 8 mL of n-hexane (Merck, 1.04374) was added to the tubes and they were centrifuged for 20 min at 5.000 rpm.The aqueous phases were extracted with 4 mL of dichloromethane (Merck, 1.06050).The organic phases were dried with a rotary evaporator (Savant, VLPI 20) at 100 rpm.Residues were collected and resuspended in 1 mL methanol for HPLC analysis.
Taxane analysis was performed using a NOVA Spher 100 Phenyl-Hexyl C18 column (250 mm × 4.6 mm, 5 µm particle size) connected with an HPLC system (Shimadzu, Japan).The mobile phase A was a mixture of distilled water, and the mobile phase B was methanol.A gradient system with A and B was used as follows: (

Statistical analysis
All experiments in this study were performed in triplicates, and data were analyzed using the GraphPad Prism (GraphPad Prism 9) software.The differences between the values for experimental groups were analyzed by analysis of variance (ordinary one-way ANOVA and two-way ANOVA).The results were expressed as the mean ± standard error (SEM).Differences were considered statistically significant at the p < 0.05 level.GraphPad Prism software was used for making graphs.

Establishment of callus and cell suspension cultures
Homogeneous cell suspension cultures (Figure 1A) were obtained from actively growing calli which were successfully established in MS medium supplemented with 2 mg L −1 2,4-D and 0.2 mg L −1 BAP after 4 weeks (Figure 1B).The growth curve of the cell suspensions was given in Figure 2. The seventeenth day of the culture was assigned as elicitation time, which is the middle of the logarithmic growth phase.

Discussion
Plant cell cultures represent an attractive alternative system for the production of natural bioactive compounds.However, a few examples of commercially successful production of secondary metabolites, such as taxanes, exist.Secondary metabolism and genetic engineering studies have been performed to increase taxane production in the main PTX producer, Taxus cell suspension cultures, in the last three decades [41,51,52].Efforts to enhance the yield of taxanes are still ongoing for Taxus spp.[12,19,22,53,54].Improvement efforts have also begun after the determination of taxanes in C. avellana.Commonly used strategies, such as medium optimization, elicitation and precursor feeding, have also been used to enhance taxane production in C. avellana [33,36,37].Because the PTX pathway has not been fully elucidated, particularly in C. avellana, elicitation and precursor feeding have been preferred options, with the former fully inducing the pathway and the latter increasing end-product production [16].
Turkey is the world's leading hazelnut producer (70-80%) and has 18 hazelnut cultivars [55,56].Hoffman [57] determined that small amounts of taxanes were isolated from the most widely cultivated cv.Tombul hazelnut.Kutlutürk [32] found that seven Turkish hazelnut cultivars accumulated taxanes in different tissues, even in their husks.Among them, cv.Kalınkara which has the highest CEPH content, was used in this study, and cell suspension cultures were established for the first time to investigate their taxane production capacity by the effect of MeJA and Phe.The use of MeJA to activate genes involved in taxane metabolic pathways is an effective strategy to increase the biotechnological production of anti-cancer compounds such as 10DAB, BACIII, CEPH and PTX [58].Sabater-Jara [59] showed that the expression levels of TXS, 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT), C-13-phenylpropanoyl-CoA transferase (BAPT), debenzoyl taxol N-benzoyl transferase (DBTNBT) genes were induced in T. media cell cultures in parallel with the increase in the amounts of 10DAB, BACIII and PTX after the addition of MeJA.The MeJA elicitor induced the PTX pathway in both Taxus spp.and C. avellana.Onrubia et al. [60] and Gallego et al. [36] showed that 100 µmol L −1 MeJA treatment increased the total taxane content almost 8 and 3-fold compared to the control in Taxus media and C. avellana, respectively.Similarly, MeJA has been shown to promote target molecules in different organisms, such as glycyrrhiza, ginkgo and Mentha [61][62][63].However, it is unclear how MeJA affects taxane production in C. avellana cv.Kalınkara.Our study found a significant increase in the total taxane production in this cultivar.In contrast to the concentrations of 100 and 300 µmol L −1 , the 200 µmol L −1 MeJA treatment resulted in an almost 8-fold increase in the total taxane content.However, the 100 and 300 µmol L −1 MeJA treatments increased the total taxane content by 3.5 and 1.5-fold, respectively.These differences in total taxane production suggest that the effect of MeJA concentration can vary depending on the genotype.The optimum MeJA concentration differed among other European cultivars [36,60].The dramatic increase in CEPH production via 200 µmol L −1 MeJA elicitation suggests that the cultivar Kalınkara might receive attention for the biotechnological production of the anti-cancer drug CEPH.To our knowledge, this is the first report of a cell suspension system in C. avellana that significantly increased CEPH production.Moreover, the production levels of 10DAB, BACIII and PTX were increased by 100 and 200 µmol L −1 MeJA treatments, similar to Taxus spp.However, 300 µmol L −1 MeJA treatment showed toxic effects and reduced the taxane production.BACIII and 10DAB were enhanced 1.66 and 1.34-fold by 100 µmol L −1 MeJA and 2.6 and 1.85-fold by 200 µmol L −1 MeJA, respectively.An increase in MeJA concentration is often correlated with an increase in taxane levels, and this enhancement is linked to the upregulation of key biosynthesis-related genes [58,59].
It has been demonstrated that adding taxane precursors to cultures is an effective strategy in C. avellana as well as Taxus spp.In addition, Phe has been reported to induce target molecules in different organisms, such as grapes, oregano and buddleia [64][65][66].Rahpeyma et al. [37] and Bemani et al. [49] determined that Phe feeding induced PTX production almost 4-fold compared with the control in C. avellana.Similarly, we observed an increase in PTX production with the treatment of 6 mmol L −1 Phe concentration.The treatment (6 mmol L −1 ) slightly increased PTX production, whereas 10DAB and BACIII levels decreased.This result indicated that 10DAB and BACIII were metabolized to end-product PTX.However, treatment with 3 µmol L −1 or 3 mmol L −1 Phe was not sufficient to promote taxane production.Variability in taxane production in cell suspension cultures is derived from genetic variation among different genotypes and cell lines, culture conditions, and time and duration of feeding [21,67].Syklowka-Baranek et al. [68] developed a taxane accumulation culture by pre-incubating amino acids such as Phe, which was subsequently elicited with MeJA.These findings suggest that the Phe precursor should be tested during this period and different culture conditions, even together with different elicitors.Overall, our results suggest that although the addition of Phe induced the formation of the end-product PTX, MeJA treatments were more effective in increasing the production of metabolites in pathways such as PTX, CEPH and taxane derivatives.Interestingly, MeJA stimulated taxane metabolites in the PTX pathway, while Phe resulted in the conversion of intermediate metabolites, such as 10DAB and BACIII, to the end-product PTX.Therefore, dual treatment with MeJA and Phe may act synergistically to produce taxanes in cv.Kalınkara.

Conclusions
In the present study, we established that C. avellana cv.Kalınkara cell suspension culture provides alternative and efficient systems for the production of PTX, CEPH and taxane derivatives.The results showed that MeJA treatments were more effective than Phe treatments for taxane production, especially CEPH.The amount of this anti-cancer drug candidate was dramatically increased by 200 µmol L −1 MeJA.Our culture system can be proposed as a prototype for the overproduction of CEPH with further optimization.

Figure 1 .
Figure 1.actively growing callus (a) and homogeneous cell suspension cultures (B) of Kalınkara hazelnut on the 30th day of cultivation in mS medium supplemented with 2 mg l −1 2,4-D and 0.2 mg l −1 Bap.

Figure 2 .
Figure 2. growth curve of Kalınkara hazelnut cell suspension cultures.the average values of three replicates are given.