Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum

Given the importance of the antitumor/antiviral lignans, podophyllotoxin and 5-methoxypodophyllotoxin, as biotechnological targets, their biosynthetic pathways were investigated in Podophyllum peltatum and Linum flavum. Entry into their pathways was established to occur via dirigent mediated coupling of E-coniferyl alcohol to afford (+)-pinoresinol; the encoding gene was cloned and the recombinant protein subsequently obtained. Radiolabeled substrate studies using partially purified enzyme preparations next revealed (+)-pinoresinol was enantiospecifically converted sequentially into (+)-lariciresinol and (-)-secoisolariciresinol via the action of an NADPH-dependent bifunctional pinoresinol/lariciresinol reductase. The resulting (-)-secoisolariciresinol was enantiospecifically dehydrogenated into (-)-matairesinol, as evidenced through the conversion of both radio- and stable isotopically labeled secoisolariciresinol into matairesinol, this being catalyzed by the NAD-dependent secoisolariciresinol dehydrogenase. (-)-Matairesinol was further hydroxylated to afford 7'-hydroxymatairesinol, this being efficiently metabolized into 5-methoxypodophyllotoxin. Thus much of the overall biosynthetic pathway to podophyllotoxin has been established, that is, from the dirigent mediated coupling of E-coniferyl alcohol to the subsequent conversions leading to 7'-hydroxymatairesinol.     

Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor

The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.     

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, dimethyl--cyclodextrin and hydroxypropyl--cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium.The complexed lignan disappeared rapidly from the culture medium, within 24h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin--d-glucoside. A maximal bioconversion rate of 0.51 mmol l^-1 suspension day^-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl--cyclodextrin complex at a final concentration of 1.35 mM.     

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l^–1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

On the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell cultures of Podophyllum hexandrum Royle

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, -glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol.     

Production of tumour-inhibitory lignans in callus cultures of Podophyllum hexandrum

Callus cultures have been established from root explants of aseptically-grown Podophyllum hexandrum seedlings. A fully defined medium based on Gamborg's B5 salts supplemented with 2/4-dichlorophenoxyacetic acid, gibberellic acid and 6-benzylaminopurine was effective for both initiation and sustained growth of callus tissue. Cultures produced anticancer lignans podophyllotoxin, 4-demethylpodophyllotoxin and podophyllotoxin 4-O-glucoside at levels similar to those found in the expiant material as assayed by high performance liquid chromatography. The relative proportions of podophyllotoxin and 4-demethyl-podophyllotoxin were markedly influenced by the presence of plant growth regulators. Particularly high levels of podophyllotoxin were associated with growth regulator induced tissue differentiation.     

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3 %) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA₃ [5 mg/l]) and higher direct somatic embryogenic frequency (89.6 %). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA₃ (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.     

Development of Linum flavum hairy root cultures for production of coniferin

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g^–1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.     

Biosynthesis of podophyllotoxin in Linum album cell cultures

Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-¹³C]3′,4′-dimethoxycinnamic acid, [2-¹³C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-¹³C]3′,4′,5′-trimethoxycinnamic acid, [2-¹³C]sinapic acid, [2-¹³C]- and [2,3-¹³C₂]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX. Electronic supplementary material to this article is available at and is accessible for authorized users. Electronic Publication     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Identification,validation, and expression of ABC transporters in Podophyllum hexandrum and their role in podophyllotoxin biosynthesis

Podophyllum hexandrum Royle is an important medicinal herb of North-Western Himalayas, and podophyllotoxin, being its major metabolite, has been used extensively in the preparation of several anticancer drugs. Podophyllotoxin accumulates in rhizomes; however, no information exists on the role of ATP-binding cassette (ABC) transporters vis-à-vis podophyllotoxin content. The present study reports identification, validation, and expression analysis of ABC transporter genes from P. hexandrum. Total 252 ABC transporter genes were identified as unigenes out of which 22 were further validated using real time qPCR in different tissues of varying podophyllotoxin content. Differential expression analysis and Pearson’s correlation coefficient revealed two candidate genes PhABC6 and PhABCIII having a positive correlation with the podophyllotoxin content. PhABCIV showed the highest expression in rhizomes (20.53-folds compared to shoots) suggesting its possible role in transport and accumulation of podophyllotoxin.     

Establishment of hairy root cultures of Linum flavum producing the lignan 5-methoxypodophyllotoxin

Hairy root cultures were induced from leaf explants of Linum flavum by infection with Agrobacterium rhizogenes. The transformed nature of tissue was confirmed by the production of opines. The cultures produced 1.5 to 3.5% of the lignan 5-methoxypodophyllotoxin (5-MPT) on a dry weight basis, which was 2 to 5 times higher than the 5-MPT content in untransformed root cultures and 5 to 12 times higher than in L. flavum cell suspensions. The 5-MPT production as a function of time was up to four times higher than that in cell suspensions.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - 5-MPT 5-methoxypodophyllotoxin - DW dry weight - GC-MS gas-chromatography coupled electron impact mass spectrometry     

Chromosome identification and karyotype analysis of Podophyllum hexandrum Roxb. ex Kunth using FISH

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).     

Effect of bacitracin on growth and monoclonal antibody production by tobacco hairy roots and cell suspensions

Hairy roots and suspended cells of transformedNicotiana tabacum were used to produce full length murine IgG₁ monoclonal antibody. The maximum amount of antibody accumulated per g dry weight in the hairy root cultures was 6.5 times that in the suspension cultures. Up to 48% of the antibody in the suspension cultures was found extracellularly, while a maximum of only 17% was recovered from the hairy root medium. The amount of assembled antibody in the root and suspension cultures was significantly reduced by intracellular and/or extracellular antibody degradation soon after the end of the exponential growth phase. Bacitracin, a polypeptide antibiotic, has been shown in previous work to prevent degradation of peptides and hormones in plant and mammalian systems. Treatment of hairy roots and cell suspensions with 100 μg/mL bacitracin was not sufficient to prevent loss of antibody from the cultures, but improved the specific growth rates by up to 53%. At concentrations of 250 μg/mL and above, bacitracin had a toxic effect on hairy roots, which may limit the application of this peptide in plant tissue culture.     

Production of hairy roots in Aconitum heterophyllum wall. using Agrobacterium rhizogenes

Summary A method for the production of hairy roots of Aconitum heterophyllum wall. is reported for the first time. Embryogenic callus cultures were successfully transformed using Agrobacterium rhizogenes strains viz. LBA 9402, LBA 9360, and A4 for the induction of hairy roots. The transgenic nature of hairy roots was confirmed by mannopine assay using paper electrophoresis. Best growth of transformed roots was obtained on 1/4 MS (Murashige and Skoog, 1962) medium with 3% sucrose. Total alkaloid (aconites) content of transformed roots was 2.96%, which was 3.75 times higher compared to 0.79% in the nontransformed (control) roots. Thin layer chromatography (TLC) analysis of the components of aconites in the transformed roots revealed the presence of heteratisine, atisine, and hetidine.     

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots

A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.     

Production of monoclonal antibodies by tobacco hairy roots

Hairy roots of tobacco (Nicotiana tabacum) were used to produce full-length murine lgG(1) monoclonal antibody. The presence of heavy (gamma) and light (kappa) chains and fully assembled antibody was verified by Western blot analysis of root extracts. Antibody levels in the biomass and medium were quantified by ELISA based on detection of gamma-kappa complexes. Antibody produced by hairy roots was fully functional as demonstrated in bacterial aggregation assays which confirmed bivalent antigen-binding capacity. Eight antibody-producing hairy root clones retained their ability to produce mouse immunoglobulin over a period of 19 months after transformation with Agrobacterium rhizogenes. For hairy roots grown in Gamborg's B5 medium, the maximum level of assembled antibody after 21-day culture in shake flasks was 18 mg L(-1) or 1.8% total soluble protein; up to 14% of the antibody was secreted into the medium. Antibody production by transgenic hairy roots had a negligible effect on growth compared with hairy roots of wild-type tobacco. Antibody accumulation was growth associated with constant specific accumulation rate at the beginning of the culture; however, degradation of antibody was significant after 14 days and the amount of assembled antibody declined. Unlike hybridoma cultures, the time course of antibody accumulation by hairy roots showed a distinctive maximum very soon after the end of exponential growth. Total antibody levels were increased by addition of nitrate, polyvinylpyrrolidone, or gelatin to the medium. Polyvinylpyrrolidone and gelatin also markedly improved extracellular antibody concentrations; with these treatments, up to 43% of the antibody present was secreted into the medium. Antibody production was tested using hairy roots grown in an air-driven bioreactor. The intracellular antibody content after 30-day bioreactor culture was similar to that measured in shake flasks; however, the final extracellular antibody level was 1.7 times higher than the maximum measured in shake flasks. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 401-415, 1997.