Application of transport engineering to promote catharanthine production in Catharanthus roseus hairy roots

Plant Cell, Tissue and Organ Culture (PCTOC) - The flowering transition is a qualitative change in the life cycle of higher plants and an important turning point in the transition from vegetative...     

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.     

Evaluation of the yeast-extract signaling pathway leading to silymarin biosynthesis in milk thistle hairy root culture

The biosynthesis of silymarin, a potent antihepatotoxic compound, from the dried fruits of Silybum marianum L. Gaertn in hairy root cultures can be stimulated by a yeast extract elicitor. These results correlated with culture time, and the biosynthesis reached a maximum of 0.47 mg g^?1 DW by 72 h after culture (2-fold higher than the control). Lipoxygenase activity and linoleic acid content were stimulated by this treatment, suggesting that the jasmonate pathway may mediate the elicitor-induced accumulation of silymarin. The H₂O₂ content increased 24 h after elicitation and did not have marked changes between 48 and 72 h. In addition, the tocopherol content (especially α- and δ-tocopherols) increased 72 h after elicitation in comparison with non-treated cultures. Ascorbate had trace changes during feeding time and was lower than the control. The antioxidant activity was assayed by the 1-1-diphenyl-2-picrylhydrazyl stable free radical method and results were calculated base on an IC₅₀ that increased upon treatment, especially 24 h after treatment, with changes related to H₂O₂ content. These observations suggested that reactive oxygen species may mediate elicitor signals to the jasmonate pathway that lead to the production of silymarin.     

Improvement of the catharanthine productivity in hairy root cultures ofCatharanthus roseus by using monosaccharides as a carbon source

Summary Sucrose, glucose, and fructose as carbon sources in culture medium were assessed in hairy root cultures ofCatharanthus roseus. The cultures preferentially consumed sucrose, resulting in about 40% (dry wt) higher growth rate. However, fructose enhanced the cathranthine yield about two-fold. The elevated yield was not seemingly ascribed to the higher osmolarity per unit weight of fructose than sucrose. A two stage culture using sucrose (1st) and fructose (2nd) improved volumetric yields of catharanthine about two-fold, i.e. 41 mg/l.     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

Enhancement of stilbene compounds and anti-inflammatory activity of methyl jasmonate and cyclodextrin elicited peanut hairy root culture

A highly stable and productive hairy root culture from peanut cultivar Tainan9 (T9-K599) was established using Agrobacterium rhizogenes strain K599 (NCPPB 2659)-mediated transformation. Valuable phenolic compounds with antioxidant activity and stilbene compounds were produced and secreted into the culture medium after elicitation with 100 µM methyl jasmonate (MeJA) and 6.87 mM cyclodextrin (CD). The antioxidant activity of the culture medium was increased to the highest Trolox equivalent antioxidant capacity (TEAC) value (28.30?±?2.70 mM Trolox/g DW) in the group treated with CD. The group co-treated with MeJA and CD exhibited the highest phenolic content, with a gallic acid equivalent (GAE) value of 10.80?±?1.00 µg gallic acid/g DW. The CuZn-SOD (CuZn superoxide dismutase) and APX (ascorbate peroxidase) antioxidant enzyme gene were up-regulated in the treatment with CD alone while the CuZn-SOD, GPX (glutathione peroxidase) and APX gene expression were down-regulated in the co-treatment with MeJA plus CD. The stilbene compounds resveratrol, trans-arachidin-1 and trans-arachidin-3 were detected by analysing the culture medium treated with CD alone and after co-treatment with MeJA and CD via HPLC. The LC-MS/MS results confirmed the presence of resveratrol, trans-arachidin-1, trans-arachidin-3, 4-Isopentadienyl-3,5,3′,4′-tetrahydroxystilbene (IPP), trans-3′-Isopentadienyl-3,5,4′-trihydroxystilbene (IPD) and arahypin-7. The results indicate that elicited peanut hairy roots can produce beneficial stilbene compounds that have antioxidant properties and anti-inflammatory activity. This peanut hairy root system could be applied as an experimental model to enhance the production of stilbene and other polyphenolic bioactive compounds.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Jasmonate-induced epoxidation of tabersonine by a cytochrome P-450 in hairy root cultures of Catharanthus roseus

Methyl jasmonate, a chemical inducer of secondary metabolism, was shown to promote tabersonine 2 biosynthesis in hairy root cultures of Catharanthus roseus. Tabersonine 6,7-epoxidase activity was detected in total protein extract of jasmonate-induced hairy root cultures using labeled 14C-tabersonine 2. This enzyme converted tabersonine 2 to lochnericine 3 by selective epoxidation at positions 6 and 7 via a reaction dependent on NADPH and molecular oxygen. Carbon monoxide, clotrimazole, miconazole, and cytochrome C were shown to be strong inhibitors of the enzyme. The activity was found in microsomes, indicating that tabersonine 6,7-epoxidase was a cytochrome P-450-dependent monooxygenase.     

Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor‐induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up‐regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90‐induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor‐induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up‐regulation of Str and Tdc. ABA‐induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO‐induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90‐induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90‐induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:994–1001, 2013     

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