Novel synthetic jasmonates as highly efficient elicitors for taxoid production by suspension cultures of Taxus chinensis

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.     

Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of l-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 M PFPJA on day 7 led to a high Tc content (38.2±0.3 mg/g) at day 21, while the Tc content was 29.3±0.3 mg/g and 34.9±0.9 mg/g with the addition of 100 M MJA and TFEJA, respectively. Quantitative structure–activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.     

Novel synthetic 2,6-dichloroisonicotinate derivatives as effective elicitors for inducing the biosynthesis of plant secondary metabolites

Two novel 2,6-dichloroisonicotinic acid (INA) derivatives [trifluoroethyl 2,6-dichloroisonicotinate (TFINA) and 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate (DPCEJ)] were chemically synthesized and evaluated by bioassay as potential elicitors for inducing the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which stably produces a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. A significant increase in Tc accumulation was observed in the presence of TFINA or DPCEJ. For example, addition of 100 μM TFINA or DPCEJ on day 7 led to a high Tc content of 21.6±2.0 or 27.7±1.0 mg g⁻¹ (on day 21), while the Tc content was 13.7±1.0 and 17.1±0.9 mg g⁻¹ for the control and that with addition of 100 μM INA, respectively. To the best of our knowledge, this is the first report on the use of synthetic INA derivatives for inducing the biosynthesis of plant secondary metabolites. The results indicate that the newly synthesized INA analogues can act as promising elicitors for secondary metabolism induction in plant cell cultures.     

Effect of the plant peptide regulator, phytosulfokine-alpha, on the growth and Taxol production from Taxus sp. suspension cultures

Phytosulfokine-alpha (PSK-alpha) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-alpha was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-alpha supplementation on production of Taxol (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-alpha supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-alpha supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-alpha added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-alpha (100 nM) and methyl jasmonate (MeJA, 100 microM) elicitation was observed, resulting in Taxol level of 35.3 +/- 2.1 mg/L or 1.83 +/- 0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture.     

水杨酸对丹参培养细胞中迷迭香酸生物合成及其相关酶的影响

迷迭香酸(RA)是丹参中一种重要的酚酸类次生代谢物。为探讨水杨酸(SA)诱导子对丹参悬浮培养细胞中RA的生物合成及其相关酶的影响,考察了SA诱导子和酪氨酸氨基转移酶(TAT)的竞争性抑制剂(AOPP)对RA合成积累量、苯丙氨酸解氨酶(PAL)和TAT活性的影响。发现在培养的第6天用浓度为6.25 mg/L的SA处理后,PAL活性在诱导后4 h出现高峰,为对照组水平的124%;RA的积累量在诱导后8 h出现峰值(5.914±0.296)mg/g。用浓度为0.1μmol/L的AOPP处理,6 h后AOPP对TAT活性影响较小(与对照组无显著差异),但明显抑制了PAL活性(为对照组水平的44%),且在PAL活性明显降低的同时RA的积累量显著减少(4.709±0.204)mg/g。进一步用0.1μmol/L AOPP和6.25 mg/L SA共处理,AOPP对PAL的抑制作用可得到一定程度的缓解,且RA的积累量较AOPP单独处理的高。表明SA可以诱导丹参悬浮培养细胞中RA积累量的增加,且在RA合成过程中PAL的限速作用比TAT明显。     

诱导子对藏红花悬浮培养细胞生产藏红花色素的影响

考察壳聚糖（chitosan）、壳寡糖（chitosanoligosaccharides,COS）、茉莉酸甲酯（methyljasmonate,MJ）、水杨酸（salicylicacid,SA）和Cu2＋等诱导子对藏红花悬浮培养细胞生长和藏红花色素合成的影响。结果表明：在实验考察浓度范围内,壳寡糖（1～500mg／L）和较低浓度壳聚糖（≤10mrdL）、MJ（≤10μmol／L）、SA（≤10μμmol／L）和Cu2＋（≤1μmoL／L）对细胞生长无显著影响;较高浓度壳聚糖（≥100mg／L）、MJ（≥100μmol／L）、SA（≥100μmoL／L）和cu“（≥10μmoL／L）显著抑制细胞生长。5种诱导子对藏红花色素合成的诱导效果不同,并且与诱导子作用浓度和添加时间有关。MJ诱导效果最好,在细胞培养第0天添加终浓度100仙moL／LMJ,藏红花色素含量（以1克干细胞计）达到28．57mg,比对照提高177．9％。其次是cu“,在细胞培养第4天添加终浓度500μmoL／LCu2＋,色素含量达到19．82mg,比对照提高108．2％。再次是壳聚糖和壳寡糖,在细胞培养第14天分别添加终质量浓度100mg／L壳聚糖和壳寡糖,色素含量分别达到18．33和17．39mg,比对照提高69．1％和69．0％。最后是SA,在细胞培养第14天添加终浓度10μmoL／LSA,色素含量达到14．65mg,比对照提高45．4％。     

Significantly improved taxuyunnanine C production in cell suspension cultures of <Emphasis Type="Italic">Taxus chinensis</Emphasis> by process intensification of repeated elicitation,sucrose feeding,and in situ adsorption

A highly efficient process intensification strategy was developed to enhance the taxuyunnanine C (Tc) production in Taxus chinensis (T. chinensis) by combining repeated elicitation with a synthesized jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA), sucrose feeding and in situ adsorption. Suspension cultures of a high Tc-producing cell line of T. chinensis were supplemented twice, on day 7 and day 12, with 100 μM DHPJA. The cultures were also supplemented by feeding 20 g sucrose/l and 100 g Ambelite XAD-7/l on day 7. This strategy had a synergistic effect on Tc synthesis, causing a significant increase in the Tc yield to 1,715.13 ± 124.12 mg/l on day 21. The yield was 3.28-fold above that produced by repeated DHPJA elicitation combined with sucrose feeding. When XAD-7 was replaced by Amberlite XAD-7 HP, the result was slightly different. The highest Tc yield of 1,728.84 ± 50.37 mg/l was achieved on day 27. The Tc yield achieved here is greater than twice the highest yield reported previously, the sole reported taxoid production yield above the gram per liter level. Given the pharmaceutical importance of taxoids, the results of this study will greatly contribute to the potential industrial production of Tc and other taxoids by plant cell cultures.     

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.     

原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产

本实验所用的中国红豆杉细胞悬浮培养体系中,云南紫杉烷c(Tc)是主要的次生代谢产物,该化合物有类神经生长因子活性,提高其产量是进一步规模化生产的前提。本研究考察了原位吸附和茉莉酸甲酯(MJA)联合调控提高Tc产量的可能性。在培养的第7天加入浓度为100μmol/L的MJA虽然会使细胞的生物量下降10%～30%,但是单位细胞内Tc含量和Tc产量均有显著提高,分别是对照的3.6和3.3倍。吸附剂XAD-7在不同时间加入对Tc的合成影响显著。在培养的第7天同时加入100μmol/L的MJA和100g/L的XAD-7会使细胞生物量增加,Tc产量显著提高。培养到第21天,Tc产量达477.4mg/L,为对照的6.3倍,为只加MJA的1.9倍,其中94%的Tc被树脂吸附。实验结果表明,在MJA诱导高表达的过程中,吸附剂XAD-7的加入使细胞内代谢产物外泌,浓度降低,减轻产物反馈抑制现象,从而大幅度提高代谢物产量,有较好的生产前景。     

Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway

Higher plants constitute one of our most important natural resources, which provide not only foodstuffs, fibers, and woods, but also many chemicals, such as flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are b…     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor)

A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.     

Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots

In different plant species, secondary metabolite biosynthesis is regulated by the phytohormone jasmonic acid (JA), which is derived by the action of lipoxygenase. In this study, we examined mono- and sesquiterpenoid accumulation and the related signal transduction pathways and biosynthetic genes in adventitious root cultures of Panax ginseng C.A. Meyer as induced by yeast extract (YE, 3 g/L), a biotic elicitor, and salicylic acid (SA, 200 μM), a signaling elicitor. The lipoxygenase (LOX) gene was highly expressed in 24 and 12 h after treatment with SA and YE. JA content was significantly increased in 24 h after SA treatment. The H₂O₂ content was the highest in 24 and 72 h after the onset of SA and YE treatment, respectively. RNA blot analysis showed that farnesyl diphosphate synthase (FPS) and isopentenyl pyrophosphate isomerase (IPPI) genes encoding enzymes of the biosynthesis of mono- and sesquiterpenoids were up-regulated by both elicitors. Farensol, isochiapin B sesquiterpenoids, champhor, and cineole monoterpenoids were highly accumulated after 24 h of SA treatment, while YE treatment induced bacchotricuneatin C, guaiazulene, isochiapin B, and p-benzoquinone sesquiterpenoid production. These results suggest that mono- and sesquiterpenoid accumulation induced by SA and YE occurs due to the IPPI and FPS expression and may be mediated by reactive oxygen species signaling and jasmonic acid signal transduction.     

Salicylic acid-induced taxol production and isopentenyl pyrophosphate biosynthesis in suspension cultures of Taxus chinensis var. mairei

The influences of salicylic acid (SA) on taxol production and isopentenyl pyrophosphate (IPP) biosynthesis pathways in suspension cultures of Taxus chinensis var. mairei were investigated by adding SA and mevastatin (MVS), a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase in the mevalonate pathway for IPP biosynthesis, into the culture systems. The cell death and taxol production were induced upon the introduction of SA, and 20mg/l was proved to be the optimal SA concentration in terms of the less damage to Taxus cells and marked activation of phenylalanine ammonia lyase (PAL). In the coexistence of SA (20mg/l) and MVS (100 nmol/l), the taxol content (1.626 mg/g dry wt) was higher than that (0.252 mg/g dry wt) of the MVS-treated system but almost equal to that (1.581 mg/g dry wt) of the SA-treated system. It is thus inferred that the activated non-mevalonate pathway should be responsible for the formation of IPP in taxol biosynthesis in the presence of SA.     

Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum

The trivalent ion of a rare earth element, lanthanum, was tested for elicitor-like effects on taxol production in suspension cultures of four different Taxus spp cells. In T. yunnanensis cell cultures, the lanthanum ion at concentrations from 1.15 to 23.0 microM stimulated taxol production. The lanthanum ion also promoted taxol excretion by the T. yunnanensis cells considerably. The maximum stimulation of taxol production was achieved by the addition of 5.8 microM La3+ to the culture during mid-log growth phase, increasing the volumetric taxol yield by nearly threefold, from 2.61+/-0.37 to 9.89+/-1.92 mg l(-1) over a 28 day culture period. At higher concentrations, i.e. 23.1 and 46.2 microM, however, the lanthanum ion caused significant growth inhibition. For the other three Taxus cell lines, namely an embryo and a leave cell of T. chinensis and a stem cell of T. chinensis marv, the addition of lanthanum ion to the culture only had a significant effect on taxol production by the T. chinensis marv stem cells, increasing the volumetric yield by about threefold to 4.69+/-0.76 mg l(-1). These results suggest that lanthanum has elicitor-like effects on secondary metabolite synthesis of plant cell cultures.     

Scale-up of centrifugal impeller bioreactor for hyperproduction of ginseng saponin and polysaccharide by high-density cultivation of panax notoginseng cells

Scale-up of a novel centrifugal impeller bioreactor (CIB) was demonstrated for production of valuable plant-specific secondary metabolites by high-density cell cultures. Initial kLa was identified to be a key factor affecting cell growth and production of ginseng saponin and polysaccharide by high-density cultivation of Panax notoginseng cells in a 3-L CIB. A high level of ginseng saponin and polysaccharide production was obtained at an initial kLa value of 30.2 h(-1). A maximum dry cell weight (DW) and production titer of ginseng saponin and polysaccharide reached 22.0 +/- 0.3, 1.5 +/- 0.1, and 2.7 +/- 0.2 g/L on day 15 with their corresponding productivity of 1140 +/- 42, 81 +/- 8, and 150 +/- 17 mg/(L.d), respectively. Based on initial kLa level, the CIB high-cell-density cultivation process was successfully scaled up from 3 L to 30 L. A maximum DW and production titer of ginseng saponin and polysaccharide in a 30-L CIB reached 25.5 +/- 0.5, 1.7 +/- 0.1, and 2.9 +/- 0.1 g/L (on day 15) at an initial kLa value of 28.7 h(-1), respectively, and their corresponding productivity was 1340 +/- 56, 91 +/- 9, and 164 +/- 15 mg/(L.d). Furthermore, by adopting a fed-batch cultivation strategy, a maximum DW and concentrations of total saponin and polysaccharide in the 30-L CIB were enhanced to 30.3 +/- 1.0, 2.1 +/- 0.1, and 3.5 +/- 0.2 g/L with their corresponding productivity of 1467 +/- 87, 102 +/- 13, and 179 +/- 18 mg/(L.d), respectively. The work suggests that the CIB may have great potential in large-scale high-density plant cell cultures for efficient production of useful secondary metabolites.