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Podophyllotoxin (PTOX), produced by Linum album, is a monolignol that participates in plant defense strategies. Our previous study established that methyl jasmonate (MeJA) significantly stimulates PTOX production in L. album cells. However; the mechanisms by which MeJA regulates PTOX biosynthesis are uncovered. In the present study, we demonstrated that MeJA induces a time-dependent hydrogen peroxide (H 2 O 2 ) and salicylic acid (SA) accumulation but reduces nitric oxide (NO) generation in L. album cells. PTOX biosynthetic genes such as PAL, CCR, CAD, and PLR were upregulated in response to MeJA exposure. Furthermore, the results of RT-qPCR revealed a positive correlation between the expression of PTOX biosynthetic genes and MeJA-induced upregulation of four miRNAs such as miR156, miR159, miR172, and miR396 at 12 h. Generally, this study revealed that MeJA mediates PTOX biosynthesis in L. album cells by inducing H 2 O 2 and SA formation, which can probably upregulate the expression level of some miRNAs and biosynthetic genes in a redox balance-dependent manner., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Previous studies have reported the functional role, biochemical features and synthesis pathway of podophyllotoxin (PTOX) in plants. In this study, we employed combined morphological and molecular techniques to identify an endophytic fungus and extract PTOX derivatives. Based on the analysis of ITS sequences and the phylogenetic tree, the isolate was classified as Penicillium herquei HGN12.1C, with a sequence identity of 98.58%. Morphologically, the HGN12.1C strain exhibits white colonies, short-branched mycelia and densely packed hyphae. Using PacBio sequencing at an average read depth of 195×, we obtained a high-quality genome for the HGN12.1C strain, which is 34.9 Mb in size, containing eight chromosomes, one mitochondrial genome and a GC content of 46.5%. Genome analysis revealed 10 genes potentially involved in PTOX biosynthesis. These genes include VdtD, Pinoresinollariciresinol reductase (PLR), Secoisolariciresinol dehydrogenase (SDH), CYP719A23, CYP71BE54, O-methyltransferase 1 (OMT1), O-methyltransferase 3 (OMT3), 2-ODD, CYP71CU and CYP82D61. Notably, the VdtD gene in fungi shares functional similarities with the DIR gene found in plants. Additionally, we identified peltatin, a PTOX derivative, in the HGN12.1C extract. Docking analysis suggests a potential role for the 2-ODD enzyme in converting yatein to deoxypodophyllotoxin. These findings offer invaluable insights into the synthesis mechanism of PTOX in fungi, shedding light on the relationship between host plants and endophytes., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Sinopodophyllum hexandrum is an alpine medicinal plant that produces the anticancer compound podophyllotoxin (PPT). Although a positive relationship between PPT content and altitude has been proved and low temperature enhances plant growth and PPT accumulation has also been revealed, the role of UV radiation in regulating growth and PPT accumulation is still unclearIn this study, morphophysiological traits, metabolites content and related genes expression were investigated by exposing S. hexandrum seedlings to treatment with UV‐B radiation.The results showed that the contents of soluble sugars and flavonoids, and the expression levels of genes involved in glycometabolism (XET and β‐1,3‐glucanase) and flavonoid biosynthesis (PAL,C4H,4CL,CHS1 and DTX41) were enhanced in response to UV‐B compared to CK. Moreover, genes involved in stress tolerance (MYB, WRKY,APX3 and EX2) were also upregulated in response to UV‐B radiation. Although the whole plant biomass exhibited slightly increased values that depended largely on root development, the contents of chlorophyll and PPT and the expression levels of genes involved in photosynthesis (matK, ndhF,rbcL and ycf5) and PPT biosynthesis (C3H,CCoAMT,CCR,CAD, DPO, PLR,SDH, CPY719A23,OMT3,CYP71CU1,OMT1and 2‐ODD) were significantly decreased in response to UV‐B compared to CK.It can be concluded that UV‐B radiation promotes soluble sugars and flavonoids accumulation, but inhibits PPT biosynthesis in S. hexandrum. [ABSTRACT FROM AUTHOR]

Highlights • Sinopodophyllum hexandrum transcriptome expression shifted with elevation. • Over 100 million high-quality reads and ca. 54 K unigenes were obtained. • Over 700 genes were differentially expressed based on elevation. • Seven podophyllotoxin-biosynthetic genes were up regulated at 3300 m above sea level. • Greater than 200 growth-promotion genes were differentially expressed with higher elevation. Abstract Sinopodophyllum hexandrum , a perennial rhizomatous herb that produces the anti-cancer metabolite podophyllotoxin (PPT), is distributed in the Himalayan region. While greater plant growth and PPT accumulation is observed at higher compared with lower elevations, the mechanism responsible for elevation-dependent growth promotion and PPT biosynthesis has not been reported. Here, the de novo sequenced transcriptome of S. hexandrum is described. When plants were grown at 3300 versus 2300 m above sea level (asl), a total of 53,691 unigenes were generated and 736 differentially expressed genes (DEGs) were observed. DEGs (357) were identified with 234 characterized genes showing greater up than down regulation, 171 versus 63, respectively. Functional annotation classified 23 DEGs involved in PPT biosynthesis, including phenylpropanoid enzymes: phenylalanine ammonia layse (PAL), cinnamyl alcohol-dehydrogenase (CAD), dirigent protein (DIR), pinoresinol-lariciresinol reductase (PLR), CYP719A23, CYP71CU1, and 2-oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD) which directly participate in PPT biosynthesis. Over 200 DEGs that participate in plant growth and development are categorized into: cell morphogenesis, bio-signaling, primary metabolism, photosynthesis/energy, transcription/polynucleotide metabolism, translation/protein synthesis, transport, and stress tolerance. This transcriptomic analysis provides insight into the mechanism that enhances plant growth and PPT accumulation in S. hexandrum under higher elevation conditions. [ABSTRACT FROM AUTHOR]

Podophyllotoxin (PPT) and its derivatives, isolated from the rhizome of Podophyllum hexandrum Royle ( P . hexandrum ), are typically used in clinical settings for anti-cancer and anti-virus treatments. Empirical studies have verified that P . hexandrum had stronger tolerance to chilling, due to involving PPT accumulation in rhizome induced by cold stress. However, the cold-adaptive mechanism and its association with PPT accumulation at a molecular level in P . hexandrum are still limited. In this study, the morpho-physiological traits related to plant growth, PPT accumulation and key gene expressions controlling PPT biosynthesis were assessed by exposing P . hexandrum seedlings to different temperatures (4 °C and 10 °C as chilling stress and 22 °C as the control). The results showed that chilling significantly increased chlorophyll content, net photosynthetic rate, stomatal conductance, and plant biomass, whereas it greatly decreased transpiration rates and intercellular CO 2 concentration. Compared to the control, the chilling treatments under 4 °C and 10 °C conditions induced a 5.00- and 3.33-fold increase in PPT contents, respectively. The mRNA expressions of six key genes were also up-regulated by chilling stresses. The findings are useful in understanding the molecular basis of P . hexandrum response to chilling. [ABSTRACT FROM AUTHOR]

Podophyllotoxin (PPT) is a plant natural product that serves as a precursor for the synthesis of many well-known chemotherapeutic drugs. The limited availability and high demand for the source plants of PPT have led to the exploration of alternative sources for this compound. In this study, we utilized the endophytic fungus Phialocephala podophylli (strain PPE7) that we isolated from the rhizomes of Podophyllum peltatum and is known to produce detectable amounts of PPT in broth culture. To date, the complete PPT biosynthetic pathway has yet to be determined in any species. Since fungi are well known for clustering genes that belong to secondary metabolite pathways, use of a fungal system for investigation of the PPT biosynthesis genes may ultimately lead to elucidation of the entire pathway. In this study, we investigated the secoisolariciresinol dehydrogenase ( SD) gene that facilitates the dehydrogenation of secoisolariciresinol to form matairesinol, a mid-pathway intermediate product in PPT biosynthesis. We utilized PCR amplification to acquire the complete SD gene sequence in PPE7 and opted to synthesize the P. peltatum SD sequence for expression. Through western blotting, we confirmed the expression of the recombinant SD (PpSD) and verified protein functionality with a bioconversion assay followed by HPLC and LC-MS analyses. Here, we report the identification of the SD gene in PPE7; this is the first report of the SD gene in an endophytic fungus. Additionally, we established the groundwork for the future expression of the complete PPT biosynthetic pathway in the heterologous host Pichia pastoris. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum Royle is known for its vast medicinal properties, particularly anticancer. It contains higher amount of podophyllotoxin (4.3 %), compared to Podophyllum peltatum (0.025 %) and other plant species; as a result, it has been used worldwide in the preparation of various drugs including anticancer, antimalarial, antiviral, antioxidant, antifungal, and so on. Currently, Etoposide (VP-16-213), Vumon® (Teniposide; VM-26), Etopophos®, Pod-Ben- 25, Condofil, Verrusol, and Warticon are available in the market. Due to highly complex synthesis and low cell culture yields of podophyllotoxin (0.3 %), the supply of raw material cannot be met due to increasing industrial demands. The knowledge on podophyllotoxin biosynthetic pathway vis-à-vis expression status of genes is fragmentary. Quantitative expression analysis of 21 pathway genes has revealed 9 genes, namely SD, PD, PCH, CM, CMT, CAD, CCR, C4H, and ADH, that showed increase in transcript abundance up to 1.4 to 23.05 folds, respectively, vis-à-vis podophyllotoxin content in roots (1.37 %) and rhizomes (3.05 %) of P. hexandrum. In silico analysis of putative cis-regulatory elements in promoter regions of overexpressed genes showed the presence of common Skn-1 motif and MBS elements in CMT, CAD, CCR, C4H, and ADH genes, thereby, suggesting their common regulation. The outcome of the study has resulted in the identification of suitable candidate genes which might be contributing to podophyllotoxin biosynthesis that can act as potential targets for any genetic intervention strategies aimed at its enhanced production. [ABSTRACT FROM AUTHOR]

Background The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway. Results 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ⩽1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRTPCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome. Conclusions The present data will help the identification of the potential genes and transcription factors involved in podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for marker discovery and conservation, which should form the foundations for future endeavors. [ABSTRACT FROM AUTHOR]

Background: The aryltetralin lignan (-)-podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (-)-podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (-)-podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes., Results: We recently established a four-step multi-enzyme cascade to convert (+)-pinoresinol into (-)-matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (-)-epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time., Conclusions: Both, (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals., (© 2021. The Author(s).)

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species ( L. flavum , L. mucronatum and L. dolomiticum ) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7- O -β-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7- O -β-glucoside.

• Sinopodophyllum hexandrum biomass increased 1.6-fold with lower temperature (15 versus 22 °C). • Low temperature increased leaf stomata and stomatal openings. • Organelle numbers increased at 15 °C. • Temperature-dependent differential gene expression associated with energy acquisition & growth. Sinopodophyllum hexandrum is a high-altitude perennial medicinal plant that produces the economically-valued anticancer metabolite, podophyllotoxin (PPT). While gene profiling under elevated altitude and low temperature conditions have identified transcriptions associated with increased PPT accumulation, changes in cell structure, with adaptive low-temperature conditions have yet to be identified. In this study, anatomical features including leaf micro- and ultra-structure were investigated by monitoring traits when S. hexandrum seedlings were grown under a cooler temperature (15 °C). Differences in leaf micro- and ultra-structure parameters including stomatal density and shape, chloroplast density as well as the abundance of starch grain, osmiophilic granule and thylakoid grana were observed with the lower temperature. Greater chlorophyll content and higher tissue amounts (e.g. leaf area, number of petioles and root size) were also observed at the cooler temperature. These anatomical and morphological changes were associated with elevated PPT levels on a dry weight and per plant basis, as well increased whole plant fresh and dry weights. To link anatomical and morphological changes with elevated PPT levels, mRNA transcripts encoding for photosynthesis, glyco-metabolism, stress response and PPT biosynthesis select genes were monitored and found to be differentially regulated at 15 versus 22 °C. These cool-temperature anatomical/morphological changes are consistent with S. hexandrum's capacity to biosynthesize and accumulate elevated PPT in cooler environments. [ABSTRACT FROM AUTHOR]

Podophyllotoxin (PTOX) is an aryl-tetralin lignan of plant origin found in some species of Podophyllum such as Dysosma versipellis, Diphylleia sinensis, and Sinopodophyllum hexandrum. Etoposide and teniposide are produced semisynthetically from PTOX and used clinically to treat several forms of cancer. As a typical representative of new drug discovery from natural products, the production of PTOX solely depends on extraction from plants, resulting in severe contradiction between supply and demand. With the advantages of unconstrained resources and eco-friendly reaction conditions, biosynthesis method has become a trend in the production of PTOX and its derivatives. In this review, we summarize the research progress of PTOX biosynthesis in plants and expound the functions of the key enzymes as well as their subcellular location. The synthetic biology for production of PTOX intermediates in a tobacco chassis is also introduced. Finally, the heterologous expression and biotransformation of PTOX in microorganisms is summarized, which sets the foundation for the efficient microbial production of PTOX using cell factories.

Dysosma pleiantha (Hance) Woodson is one of the endangered traditional Chinese medicinal herbs, highly valued for its medicinal properties by Taiwan's mountain tribes. The present study aims to develop an efficient protocol for callus biomass by optimizing suitable culture medium, carbon source culture condition, and enhanced production of pharmaceutically important podophyllotoxin, kaempferol, and quercetin from callus culture of D. pleiantha under the influence of different additives. Best callus induction was achieved in Gamborg's medium (B5) with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) along with 0.2 mg/L kinetin under dark condition. Tender leaves of D. pleiantha showed the maximum of 86% callus induction among the different explants tested. Highest leaf callus proliferation was noted in B5 medium with 1 mg/L 2,4-D incubated under complete darkness. In addition, it was found that B5 medium with 1 mg/L 2,4-D along with 2 g/L peptone produced more leaf callus biomass and enhanced production of podophyllotoxin (16.3-fold), kaempferol (12.39-fold), and quercetin (5.03-fold) compared to control. Therefore, D. pleiantha callogenesis can provide an alternative source for enhanced production of secondary compounds regardless of the exploitation of its natural plant population., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

(-)-Podophyllotoxin is one of the most potent microtubule depolymerizing agents and has served as an important lead compound in antineoplastic drug discovery. Reported here is a short chemoenzymatic total synthesis of (-)-podophyllotoxin and related aryltetralin lignans. Vital to this approach is the use of an enzymatic oxidative C-C coupling reaction to construct the tetracyclic core of the natural product in a diastereoselective fashion. This strategy allows gram-scale access to (-)-deoxypodophyllotoxin and is readily adaptable to the preparation of related aryltetralin lignans., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

A complementary study including design of mechanistic probes, biochemical assays, model analysis, and liquid chromatography coupled mass spectrometry was conducted to establish the reaction mechanism for a nonheme iron enzyme catalyzed (-)-podophyllotoxin formation. Our results indicate that the originally proposed hydroxylated intermediate is unlikely to be involved in this reaction. Instead, the formation of benzylic radical/carbocation intermediate can be utilized to trigger the C-C bond formation to construct the C-ring of (-)-podophyllotoxin.

Secondary metabolites from numerous plant sources have been developed as anti- cancer reagents and compounds such as resveratrol, podophyllotoxin and zerumbone are of particular importance in this regard. Since their de novo chemical synthesis is both arduous and commercially expensive, there has been an impetus to develop viable, biotechnological methods of production. Accordingly, this review focuses on the recent developments in the field, highlighting the use of micropropagation, cell suspension cultures, callus cultures, hairy root cultures, recombinant microbes and genetically modified higher plants. Optimization of media and culture conditions, precursor feeding, immobilization and the use of chemical or physical elicitation in various protocols has led to an increase in resveratrol and podophyllotoxin production. Heterologous gene transformation of higher plants with stilbene synthase derived from Arachis hypogaea or Vitis vinifera lead to resveratrol production with the concomitant increase in resistance to plant pathogens. Interestingly, genetic transformation of Podophyllum hexandrum and Linum flavum with Agrobacterium rhizogenes resulted in Ri-T-DNA gene(s)-mediated enhancement of podophyllotoxin production. Zerumbone yields from tissue cultured plantlets or from suspension cultures are generally low and these methods require further optimization. In microbes lacking the native resveratrol or zerumbone biosynthesis pathway, metabolic engineering required not only the introduction of several genes of the pathway, but also precursor feeding and optimization of gene expression to increase their production. Data pertaining to safety and toxicity testing are needed prior to use of these sources of anti-cancer compounds in therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Plant tissue cultures are a potential source of secondary metabolites. However, their production, when compared with intact plants, is usually lower. Phenylalanine, a biogenetic precursor of podophyllotoxin, was used to stimulate podophyllotoxin production in callus and suspension cultures of Juniperus virginiana L. The best phenylalanine effect on podophyllotoxin production was manifested in three-years-old callus cultures after a 21-days application of a 10 mmol/L concentration. A podophyllotoxin content of 0.15 mg/g DW was determined, which was about 400% higher in comparison with the control. The maximum content (0.48 mg/g DW) in newly derived suspension cultures (the 4' passage) was induced by 14-days application of a I mmol/L concentration; this was about 243% higher than the control. In one-year-old suspension cultures the highest podophyllotoxin content (0.56 mg/g DW) was recorded also after 14-days application of a I mmol/L concentration; this was about 211% higher than in the control cultures.

Podophyllum species (Podophyllum hexandrum Royle and Podophyllum peltatum) are a major source of deriving anticancer drugs from their major chemical constituent, podophyllotoxin. However, information lacks on regulatory components of podophyllotoxin biosynthesis; therefore, different classes of transcription factors were identified through mining transcriptomes of Podophyllum species and validated through qRT-PCR analysis vis-à-vis podophyllotoxin contents in different tissues/organs of Podophyllum hexandrum. A total of 82, 278, 70, and 90 transcripts were identified in shoots and 89, 273, 72, and 91 transcripts in rhizomes of P. hexandrum transcriptome; 70, 268, 48, and 92 transcripts were in shoots and 58, 245, 41, and 85 transcripts in rhizomes of P. peltatum transcriptome corresponding to bZIP, MYB, WRKY, and bHLH families of transcription factors, which have been shown in regulating biosynthesis of secondary metabolites. Two unique transcripts encoding bHLH and MYB/SANT TFs in shoots of P. peltatum (medp_podpe_41091 and medp_podpe_2547) and bZIP and MYB TFs in rhizomes of P. hexandrum (medp_podhe_163581 and medp_podhe_147614) correlated with podophyllotoxin content. Quantification of podophyllotoxin and comparative expression analysis between high (2.51 %) versus low (0.59) podophyllotoxin content accessions revealed 0.04 to ~16-folds increase in transcripts of transcription factors, thereby further supporting the association of identified transcription factors with podophyllotoxin content. bZIP TF showed the highest transcript abundance (19.60-folds) in P. hexandrum rhizomes (2.51 % podophyllotoxin) compared to shoots (0.01 %). In silico analysis of putative promoter regions of pathway genes in other plant species revealed the presence of sequence elements for MYB and WRKY transcription factors, thereby suggesting their role in controlling the production of podophyllotoxin. A repertoire of additional transcription factors has been provided, which can be functionally validated and used in designing a suitable genetic intervention strategy towards enhanced production of podophyllotoxin.

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin., (Copyright © 2015, American Association for the Advancement of Science.)

The Linum thracicum ssp. thracicum cell lines developed in this study are a feasible source for the sustainable production of podophyllotoxin, a lignan with an aryltetralin skeleton that is used for the manufacture of the chemotherapeutic drugs etopophos and teniposide. We used mass spectrometry to confirm the presence of the aryltetralin lignan in the thracian flax cell cultures. Next, we explored how changes in the culture medium influenced the podophyllotoxin content. Out of six developed cell lines, four were selected for further experiments and challenged with elicitors. The selected cell lines clustered into two groups: developed in full strength medium (Li) vs developed in half strength medium (HS). While podophyllotoxin production in the Li cell lines was boosted by 80% upon administration of the elicitor methyl jasmonate, the HS lines produced high amounts of the target metabolite triggered by reduced concentration of nutrients and were only slightly influenced by the elicitor.

Background: Sinopodophyllum hexandrum is an endangered medicinal herb, which is commonly present in elevations ranging between 2,400-4,500 m and is sensitive to temperature. Medicinal property of the species is attributed to the presence of podophyllotoxin in the rhizome tissue. The present work analyzed transcriptome of rhizome tissue of S. hexandrum exposed to 15°C and 25°C to understand the temperature mediated molecular responses including those associated with podophyllotoxin biosynthesis., Results: Deep sequencing of transcriptome with an average coverage of 88.34X yielded 60,089 assembled transcript sequences representing 20,387 unique genes having homology to known genes. Fragments per kilobase of exon per million fragments mapped (FPKM) based expression analysis revealed genes related to growth and development were over-expressed at 15°C, whereas genes involved in stress response were over-expressed at 25°C. There was a decreasing trend of podophyllotoxin accumulation at 25°C; data was well supported by the expression of corresponding genes of the pathway. FPKM data was validated by quantitative real-time polymerase chain reaction data using a total of thirty four genes and a positive correlation between the two platforms of gene expression was obtained. Also, detailed analyses yielded cytochrome P450s, methyltransferases and glycosyltransferases which could be the potential candidate hitherto unidentified genes of podophyllotoxin biosynthesis pathway., Conclusions: The present work revealed temperature responsive transcriptome of S. hexandrum on Illumina platform. Data suggested expression of genes for growth and development and podophyllotoxin biosynthesis at 15°C, and prevalence of those associated with stress response at 25°C.

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.

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Podophyllotoxin (PTOX) is a naturally occurring phenolic compound isolated as an active anti-tumor agent. The stimulatory influence of light on the formation of phenolic compounds has been reported, but the molecular mechanism underlying the effect of light on the expression of genes involved in phenolic biosynthesis, especially of lignans, is still not fully understood. A series of experiments was carried out using ordinary fluorescent lamps to study the influence of light irradiation on growth and PTOX accumulation in Linum album cell cultures by varying the type of light and periods of exposure. The biosynthesis of PTOX was variably affected according to the quality of light. The enhancing effects of red light on PTOX production was correlated with increased activities of the enzyme phenylalanine ammonia-lyase (PAL), and the expression of some key genes involved in the biosynthesis of this compound, including the PAL gene itself and the cinnamoyl-CoA reductase (CCR) gene. Blue light was found to have similar effects but mainly on the expression level of CCR and pinoresinol lariciresinol reductase (PLR) genes., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [140μgg(-1) dry weight (DW) of the L. album cell culture] which is seven-fold greater than the untreated control, while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol, instead of PTOX, up to 365μgg(-1) DW, which was 8.8-fold greater than the control. Quantitative PCR analyses showed that expression of the enzyme genes responsible for the PTOX biosynthesis cascade, such as pinoresinol-lariciresinol reductase (PLR), phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) genes, were also up-regulated in a fungal extract-selective fashion. These results provide evidence that the fungal extracts used in this study differentially increase the production of PTOX or larisiresinol via the up-regulation of the genes in lignan biosynthesis in L. album cell cultures, and suggest that such selective actions of fungal elicitors on the lignan synthesis will lead to more efficient metabolic engineering-based production of PTOX and other beneficial lignans using L. album cell cultures., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

This study reports an appreciable yield of podophyllotoxin (PDT) in P. hexandrum plants grown ex situ under polyhouse conditions of a temperate locale. The PDT content of below-ground parts was affected by both plant age and growth period. However, only the effect of plant age on PDT content was significant. Thus, the highest amounts of PDT were recorded in the below-ground parts of 2-year-old plants harvested during the late-growth period (LGP). High total soluble sugars in the below-ground parts during the early growth period (EGP) and the highest nitrate and nitrate reductase in the leaves of 2-year-old plants during the peak-growth period (PGP) indicated higher mobilization and assimilation of starch and nitrate. Probably the surplus carbon and nitrogen gained during the PGP were diverted from aerial parts to below-ground parts during the LGP and in turn contributed to the synthesis of higher amounts of PDT. This study shows that commercial cultivation of P. hexandrum is possible under ex situ temperate conditions.

The aryltetralin lignan deoxypodophyllotoxin is much more widespread in the plant kingdom than podophyllotoxin. The latter serves as a starting compound for the production of cytostatic drugs like etoposide. A better insight into the occurrence of deoxypodophyllotoxin combined with detailed knowledge of its biosynthestic pathway(s) may help to develop alternative sources for podophyllotoxin. Using HPLC combined with electrospray tandem mass spectrometry and NMR spectroscopy techniques, we found nine lignans and five related structures in roots of Anthriscus sylvestris (L.) Hoffm. (Apiaceae), a common wild plant in temperate regions of the world. Podophyllotoxone, deoxypodophyllotoxin, yatein, anhydropodorhizol, 1-(3'-methoxy-4',5'-methylenedioxyphenyl)1-ξ-methoxy-2-propene, and 2-butenoic acid, 2-methyl-4-[[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1-yl ester, (2Z)- were the major compounds. α-Peltatin, podophyllotoxin, β-peltatin, isopicropodophyllone, β-peltatin-a-methylether, (Z)-2-angeloyloxymethyl-2-butenoic acid, anthriscinol methylether, and anthriscrusin were present in lower concentrations. α-Peltatin, β-peltatin, isopicropodophyllone, podophyllotoxone, and β-peltatin-a-methylether have not been previously reported to be present in A. sylvestris. Based on our findings we propose a hypothetical biosynthetic pathway of aryltetralin lignans in A. sylvestris., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Podophyllum hexandrum, known for its diversified clinical importance particularly for antineoplastic activity and valuable source for biological protection against high doses of radiation, has its unique position in the plant kingdom. Detailed understanding of mechanism and opportunity of chemical manipulations has amplified the scope of its bioactivity. Podophyllotoxin, the major active principle of this plant, has passed through various structural deviations with the basic aim of making the end product clinically more effective with minimal toxicity. However, over exploitation and limited growth has categorized this plant under endangered species. Depending upon the geographical variations, different species and subspecies of this plant have been explored. Morphological variations and quantitative differences in active principles are the major concern of its unstable medicinal value in whole and semifractionated preparations. The current review has addressed the issues related to the genetic diversity of P. hexandrum, extrinsic and intrinsic stresses responsible for its diversified nature, chemical modifications to enhance its multitasking bioactivity, and efforts for its cultivation and production of important metabolites to avoid collection of wild species due to its critically endangered nature.

Podophyllotoxin, one of the well-known naturally occurring aryltetralin lignans, has been used as the lead-compound for the preparation of potent anticancer agents, such as etoposide, teniposide, and etopophos. In our previous review, we described the advances of podophyllotoxin derivatives from 2003 and 2007. In recent years, an increased number of interesting research work has been carried out on the podophyllotoxins. As a continuation, the present review summarizes and highlights the update advances of podophyllotoxin derivatives from 2008 and 2010 in regard to semisynthesis, biosynthesis, biological activities, mode of action and structure-biological activity relationship.

Malignant diseases are the second mortality cause within the human population. Despite the serious progress in establishing and introduction of novel specifically targeted drugs the therapy of these diseases remains severe medical and social problem. Some of the most effective cancer treatments to date are natural products or compounds derived from plant products. Isolation of anticancer pharmaceuticals from plants is difficult due to their extremely low concentrations. The industry currently lacks sufficient methods for producing all of the desired plant-derived pharmaceutical molecules. Some substances can only be isolated from extremely rare plants. Plant cell cultures are an attractive alternative source to whole plant for the production of high-value secondary metabolites. The biotechnological method offers a quick and efficient method for producing these high-value medical compounds in cultivated cells. Due to the pharmaceutical importance and the low content in the plants the present review focuses on discovery and alternative production systems for anticancer lignans--aryltetralin and arylnaphthalene lignans. The aim is to focus on recent progress of in vitro production of anticancer lignans, together with structure elucidation, the methods of increasing the levels of desired substances in plant cell and tissue cultures in general. Experience of different authors, working worldwide on plant biotechnology, has been discussed to show positive results in experiments.

Podophyllotoxin (PTOX) is an extremely important plant-derived natural product, of which derivatives, like etoposide and teniposide, have been widely applied in therapies for cancers and venereal wart. A durable, intense plant extraction of podophyllotoxin posed a severe pressure on wild resources; researchers consequently sought to explore new sources, like cultivation, plant cell or organ culture, and chemical synthesis. Understanding biosynthesis of PTOX is one of the basic necessary steps for standard cultivation of medicinal plants and metabolite engineering. An important progress has been made in this field during the last two decades, particularly in the last ten years. Although a number of reviews concerning the related topic have existed, we specifically deal with biosynthesis of podophyllotoxin with an emphasis on the literatures of the past decade, highlighting characterization of genes encoding synthetic enzymes and down-stream metabolism of PTOX. The present review focuses on several key biosynthesis processes, important metabolites, function of related enzymes, and characterization of cDNA encoding the enzymes. Finally, the author proposed a hypothetical biosynthetic scheme of podophyllotoxin and perspectives.

Treatment of Linum album cell cultures with 10 μM salicylic acid (SA) for 3 days improved podophyllotoxin (PTOX) production up to 333 μg/g dry weight (DW): over three times that of the control cultures. qPCR analyses showed that in SA-treated cells, the expression of the genes coding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD), all involved in the first steps of PTOX biosynthesis, also increased reaching a peak 8-12 h after the treatment. Expression of the pinoresinol-lariciresinol reductase gene (PLR), which is involved in one of the last biosynthetic steps, was not affected by SA. The selective action of SA on these genes can be applied to control the biotechnological production of this anticancer agent.

A novel biotransformation process of podophyllotoxin (1) to produce picropodophyllotoxin (2) and podophyllic acid (3) was developed in this work. Eight bacteria which could modify the structure of podophyllotoxin were screened out from the tested fourteen bacteria. The highest conversion of podophyllotoxin (i.e., 70.2 +/- 8.0%) was obtained when Pseudomonas aeruginosa CCTCC AB93066 was used as biocatalyst, so P. aeruginosa was selected as a typical biocatalyst in the following study. Product (2) and (3) were separated through D312 macroporous resin and sephadex LH-20 gel column chromatograph. On the basis of (1)H NMR, (13)C NMR, ESI-MS and Elemental Analysis, product (2) and (3) were identified as picropodophyllotoxin (2) and podophyllic acid (3), respectively. This suggested the site-specific isomerization and hydrolization of podophyllotoxin occurred during its biotransformation process by P. aeruginosa. For the first time, podophyllotoxin was biotransformed into its hydrolytic derivate (i.e., podophyllic acid).

This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3+/-3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3+/-3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2+/-0.1%). 1g/l of NaCl was the best for podophyllic acid production (i.e. 47.6+/-4.0mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9+/-12.3mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0+/-2.1mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid.

Podophyllotoxin is a plant-derived compound found in Podophyllum sp. that is used to produce semi-synthetic anticancer pharmaceuticals such as etoposide, teniposide, and etoposide phosphate. This chapter describes the role of biotechnology to produce podophyllotoxin and our attempts to domesticate Podophyllum peltatum L., also known as the American mayapple. The domestication research on mayapple included surveys of the natural population, identification of high yielding genotypes, propagation, cultivation, sustainable harvest procedures and the development of protocols for in vitro germplasm bank.

The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.

Cell suspension cultures of Linum album were developed from internode portions of in vitro germinated plant in Gamborg's B5 medium supplemented with 0.4 mg naphthalene acetic acid/l. The highest biomass was 8.5 g/l with podophyllotoxin and 6-methoxypodophyllotoxin at 29 and 1.9 mg/l, respectively after 12 d cultivation. Co-cultures of L. album cells with axenically cultivable arbuscular mycorrhiza-like fungi, Piriformospora indica and Sebacina vermifera, were established for the first time. These enhanced podophyllotoxin and 6-methoxypodophyllotoxin production by about four- and eight-fold, respectively, along with a 20% increase in biomass compared to the control cultures.

Callus and suspension cultures of Linum album as an Iranian species for synthesis of aryltetralin lactone were used. After cultivation period; we can isolate and characterize podophyllotoxin as glycosides compounds. Maximal product yield is 0.5% of the dry weight in dark grown cultures.

The morphological features of Podophyllum hexandrum Royle, a 'critically endangered' medicinal herb and a source of podophyllotoxin, were studied in populations growing in different parts of the Kumaun region of the Indian Central Himalaya. Plant growth performance in terms of biomass accumulation and podopyllotoxin levels in the rhizomes collected from eleven natural populations (P1 to P11, altitude ranging from 2740 to 3350 m) were analyzed. Morphological features, e. g., plant height, stem diameter and leaf area were, in general, negatively correlated with an increase in the altitude. Maximum aboveground (8.46 g/individual) and belowground (48.18 g/individual) biomass values were recorded from a population (P9) at the lowest altitude (2740 m) and, in general, the species was found to perform better at the lower altitudes. The podophyllotoxin content of rhizomes ranged between 0.36-1.08% (on dry wt. basis) in different populations, and a positive correlation was observed between podophyllotoxin content and an increase in the altitude.

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 microM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5'-demethoxy-MPTOX (5'-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5'-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and beta-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69+/-1.45 mg/g dry weight and 1.11+/-0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49+/-0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.

Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.

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.