Plant cell and organ cultures as a source of phytochemicals

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/67430Plant cell and organ cultures constitute a promising platform for the production of numerous valuable secondary compounds. Currently, in vitro culture techniques involve both empirical and rational approaches as suitable strategies to condition high metabolite production and establish competitive plant cell-based bioprocesses. In this context, we have developed hairy root cultures of Panax ginseng, and engineered hairy root cultures of Duboisia, Datura metel and Hyoscyamus spp and plant cell cultures of Centella asiatica and Taxus spp. This chapter describes our work on the development of two different biotechnological systems to improve taxol production in cell suspension cultures of Taxus spp and ginsenoside production in hairy root cultures of Panax ginseng

Biotechnological production of taxanes: a molecular approach

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/46988Plant cell cultures constitute a promise for the production of a high number of phytochemicals, although the majority of bioprocesses that have been developed so far have not resulted commercially successful. An overview indicates that most of the research carried out until now is of the empirical type. For this reason, there is a need for a rational approach to the molecular and cellular basis of metabolic pathways and their regulation in order to stimulate future advances. The empirical investigations are based on the optimization of the culture system, exclusively considering input factors such as the selection of cellular lines, type and parameters of culture, bioreactor design and elicitor addition, and output factors such as cellular growth, the uptake system of nutrients, production and yield. In a rational approach towards the elucidation of taxol and related taxane production, our group has studied the relationship between the taxane profile and production and the expression of genes codifying for enzymes that participate in early, intermediate and late steps of their biosynthesis in elicited Taxus spp cell cultures. Our results show that elicitors induce a dramatic reprogramming of gene expression in Taxus cell cultures, which likely accounts for the enhanced production of taxol and related taxanes and we have also determined some genes that control the main flux limiting steps. The application of metabolic engineering techniques for the production of taxol and taxanes of interest is also discussed

Gene expression pattern and taxane biosynthesis in a cell suspension culture of Taxus baccata L. subjected to light and a phenylalanine ammonia lyase (PAL) inhibitor

Taxus baccata L. cell culture is a promising commercial method for the production of taxanes with anti-cancer activities. In the present study, a T. baccata cell suspension culture was exposed to white light and 2-aminoindan- 2-phosphonic acid (AIP), a phenylalanine ammonia lyase (PAL) inhibitor, and the effects of this treatment on cell growth, PAL activity, total phenol content (TPC), total flavonoid content (TFC), taxane production and the expression of some key taxane biosynthetic genes (DXS, GGPPS, T13OH, BAPT, DBTNBT) as well as the PAL were studied. Light reduced cell growth, whereas AIP slightly improved it. Light increased PAL activity up to 2.7-fold relative to darkness. The highest TPC (24.89 mg GAE/g DW) and TFC (66.94 mg RUE/g DW) were observed in cultures treated with light and AIP. Light treatment also resulted in the maximum content of total taxanes (154.78 μg/g DW), increasing extracellular paclitaxel and cephalomannin (3.3-fold) and intracellular 10-deacetyl paclitaxel (2.5-fold). Light significantly increased the expression level of PAL, DBTNBT, BAPT, and T13αOH genes, whereas it had no effect on the expression of DXS, a gene active at the beginning of the taxane biosynthetic pathway. AIP had no significant effect on the expression of the target genes. In conclusion, the light-induced activation of PAL transcription and altered expression of relevant biosynthetic genes reduced cell growth and increased the content of total phenolic compounds and taxanes. These findings can be applied to improve taxane production in controlled cultures and bioreactors

Genetic transformation of Artemisia carvifolia Buch with rol genes enhances artemisinin accumulation

The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.010.8% DW). There is therefore an urgent need to design new strate- gies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: arte- misinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin con- tent in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism

Enhanced artemisinin yield by expression of rol genes in artemisia annua

Background: Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug. Methods: In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined. Results: Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant. Conclusion: Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), Generalitat de Catalunya (2014SGR215) and European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1-Senescyt-2014) and a grant from UTEQ (UTEQAmbiental-9-FCAmb-IFOR-2014-FOCICYT002)

Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions

More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-β-cyclodextrins (β-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with β - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + β - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells. Keywords: Taxus baccata; cell cultures; coronatine; gene expression; lipid droplets; paclitaxel; taxane accumulation

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Summary Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrolconverting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yield

Tailoring tobacco hairy root metabolism for the production of stilbenes

Tobacco hairy root (HR) cultures, which have been widely used for the heterologous production of target compounds, have an innate capacity to bioconvert exogenous t-resveratrol (t-R) into t-piceatannol (t-Pn) and t-pterostilbene (t-Pt). We established genetically engineered HR carrying the gene encoding stilbene synthase (STS) from Vitis vinifera and/or the transcription factor (TF) AtMYB12 from Arabidopsis thaliana, in order to generate a holistic response in the phenylpropanoid pathway and coordinate the up-regulation of multiple metabolic steps. Additionally, an artificial microRNA for chalcone synthase (amiRNA CHS) was utilized to arrest the normal flux through the endogenous chalcone synthase (CHS) enzyme, which would otherwise compete for precursors with the STS enzyme imported for the flux deviation. The transgenic HR were able to biosynthesize the target stilbenes, achieving a production of 40 μg L-1 of t-R, which was partially metabolized into t-Pn and t-Pt (up to 2.2 μg L-1 and 86.4 μg L-1, respectively), as well as its glucoside piceid (up to 339.7 μg L-1). Major metabolic perturbations were caused by the TF AtMYB12, affecting both primary and secondary metabolism, which confirms the complexity of biotechnological systems based on seed plant in vitro cultures for the heterologous production of high-value molecules

Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance

Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds - commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity