Search

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., ((c) 2006 Wiley Periodicals, Inc.)

Recently novel synthetic jasmonate derivatives were shown to have very powerful stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells (Biotech Bioeng, 86:595; ibid. 86: 809, 2004). To provide an insight into the elicitation mechanism of the newly synthesized elicitors, by taking 2-hydroxyethyl jasmonate (HEJ, at 100 microM) as a typical example, in this work the defense signals were detected and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in cell cultures of T. chinensis. The oxidative burst (induced H2O2 production) was confirmed, and the induction of lipoxygenase (LOX) activity and intracellular jasmonate acid (JA) synthesis was found. The gene transcription of geranylgeranyl diphosphate synthase (GGPPs) and taxa-4(5),11(12)-diene synthase (TS) was up-regulated by HEJ elicitation compared to control. Inhibition of JA biosynthesis by a putative LOX inhibitor, ibuprofen (IBU), effectively depressed the HEJ-induced up-regulation of GGPPs and TS gene transcription levels. In contrast, the inhibition of H2O2 production by membrane NADPH oxidase inhibitor, diphenylene iodonium (DPI), did not affect the transcriptions of those genes. For the HEJ-induced Tc production, it was suppressed with addition of DPI or IBU. The results suggest that both H2O2 and JA signals were involved in HEJ-induced Tc biosynthesis, and JA mediated the induction of GGPPs and TS genes expression, but H2O2 was not essential to activate them. Finally, a signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed., (Copyright 2006 Wiley Periodicals, Inc.)

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., ((c) 2005 Wiley Periodicals, Inc.)

Paclitaxel and baccatin III-producing cells of Taxus baccata were immobilized within Ca(2+)-alginate beads. Under established optimum conditions for the biosynthesis of both taxanes, the yields of paclitaxel and baccatin III in shake-flask cultures of free cells increased by factors of up to 3 and 2, respectively, in the corresponding cultures of immobilized cells. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities when both free and immobilized cells were grown in the same optimum conditions in three different bioreactor types (Stirred, Airlift, and Wave) running for 24 days in a batch mode and with the system optimized in each case, there was a considerable increase in the yields of paclitaxel and baccatin III. Among the reactors, the Stirred bioreactor was the most efficient in promoting immobilized cell production of paclitaxel, giving a content of 43.43 mg.L(-1) at 16 days of culture, equivalent to a rate of 2.71 mg.L(-1).day(-1). To our knowledge, the paclitaxel productivity obtained in this study is one of the highest reported so far by academic laboratories for Taxus species cultures in bioreactors.

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., (Copyright 2004 Wiley Periodicals, Inc.)

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., (Copyright 2004 Wiley Periodicals, Inc.)