Optimization of the fermentation media, mathematical modeling, and enhancement of paclitaxel production by Alternaria alternata after elicitation with pectin

Alternaria alternata fungus is a potent paclitaxel producer isolated from Corylus avellana. Themajor challenge is the lack of optimized media for endophytic fungi productivity. In the effort tomaximize the production of taxoids by A. alternata, several fermentation conditions, including pH(pH 4.0–7.0), different types and concentrations of carbon (fructose, glucose, sucrose, mannitol,sorbitol, and malt extract), and nitrogen (urea, ammonium nitrate, potassium nitrate, ammoniumphosphate, and ammonium sulfate) were applied step by step. Based on the results, A. alternata in amedium containing sucrose 5% (w/v) and ammonium phosphate 2.5 mM at pH 6.0 showed a rapid andsustainable growth rate, the highest paclitaxel yield (94.8 μg gFW−1 vs 2.8 μg gFW− 1 in controls), andthe maximum content of amino acids. Additionally, the effect of pectin was evaluated on fungus, andmycelia harvested. Pectin significantly enhanced the growth and taxoid yield on day 21 (respectively171% and 116% of their corresponding on day 7). The results were checked out by mathematicalmodeling as well. Accordingly, these findings suggest a low-cost, eco-friendly, and easy-to-produceapproach with excellent biotechnological potential for the industrial manufacture of taxoids.</p

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

Perfluorodecalin-supported system enhances taxane production in hairy root cultures of Taxus x media var. Hicksii carrying a taxadiene synthase transgene

Enhanced taxane production was observed in a hybrid, two liquid phases containing, cultures of Taxus x media var. Hicksii hairy root carrying a taxadiene synthase transgene, supported with liquid perfluorodecalin (PFD) in degassed or aerated form. The hairy root cultures were elicited with methyl jasmonate (MJ, 100 μM) or coronatine (COR, 1 μM), and fed with sucrose and l-phenylalanine. The root growth was not stimulated by PFD addition, irrespective of the day of its application (day 0 and 14). However, in the cultures elicited with MJ and performed in the presence of PFD the final root biomass accumulation was higher than in cultures performed without PFD while the opposite effect was observed in cultures supplemented with COR. The highest paclitaxel content in root biomass was determined at the end of the cultures elicited with MJ and supplemented with PFD-degassed at day 0 or 14, 1,440.8 and 1,432.5 μg g−1 DW, respectively. The highest total (i.e. intracellular + extracellular: both in aqueous and PFD phases) paclitaxel yield in flasks (149.15 μg flask−1) was noted after the application of PFD-degassed at day 14. The other taxane detected was baccatin III, only in the root biomass, with the highest content (76.9 μg g−1 DW) observed under COR treatment. Although COR stimulated paclitaxel production with less efficiency than MJ, it resulted in higher paclitaxel excretion to the liquid phases of culture medium and PFD

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

Impact of Elicitation on Plant Antioxidants Production in Taxus Cell Cultures

Elicited cell cultures of Taxus spp. are successfully used as sustainable biotechnological production systems of the anticancer drug paclitaxel, but the effect of the induced metabolomic changes on the synthesis of other bioactive compounds by elicitation has been scarcely studied. In this work, a powerful combinatorial approach based on elicitation and untargeted metabolomics was applied to unravel and characterize the effects of the elicitors 1 M of coronatine (COR) or 150 M of salicylic acid (SA) on phenolic biosynthesis in Taxus baccata cell suspensions. Differential effects on cell growth and the phenylpropanoid biosynthetic pathway were observed. Untargeted metabolomics analysis revealed a total of 83 phenolic compounds, mainly flavonoids, phenolic acids, lignans, and stilbenes. The application of multivariate statistics identified the metabolite markers attributed to elicitation over time: up to 34 compounds at 8 days, 41 for 16 days, and 36 after 24 days of culture. The most notable metabolic changes in phenolic metabolism occurred after 8 days of COR and 16 days of SA elicitation. Besides demonstrating the significant and differential impact of elicitation treatments on the metabolic fingerprint of T. baccata cell suspensions, the results indicate that Taxus ssp. biofactories may potentially supply not only taxanes but also valuable phenolic antioxidants, in an efficient optimization of resources

Biotic Elicitors in Adventitious and Hairy Root Cultures: A Review from 2010 to 2022

One of the aims of plant in vitro culture is to produce secondary plant metabolites using plant cells and organ cultures, such as cell suspensions, adventitious, and hairy roots (among others). In cases where the biosynthesis of a compound in the plant is restricted to a specific organ, unorganized systems, such as plant cell cultures, are sometimes unsuitable for biosynthesis. Then, its production is based on the establishment of organ cultures such as roots or aerial shoots. To increase the production in these biotechnological systems, elicitors have been used for years as a useful tool since they activate secondary biosynthetic pathways that control the flow of carbon to obtain different plant compounds. One important biotechnological system for the production of plant secondary metabolites or phytochemicals is root culture. Plant roots have a very active metabo-lism and can biosynthesize a large number of secondary compounds in an exclusive way. Some of these compounds, such as tropane alkaloids, ajmalicine, ginsenosides, etc., can also be biosyn-thesized in undifferentiated systems, such as cell cultures. In some cases, cell differentiation and organ formation is necessary to produce the bioactive compounds. This review analyses the biotic elicitors most frequently used in adventitious and hairy root cultures from 2010 to 2022, focusing on the plant species, the target secondary metabolite, the elicitor and its concentration, and the yield/productivity of the target compounds obtained. With this overview, it may be easier to work with elicitors in in vitro root cultures and help understand why some are more effective than others

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

Exploring the interplay between metabolic pathways and taxane production in elicited Taxus baccata cell suspensions

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.Agencia Estatal de Investigación | Ref. PID2020-113438RB-I00Agència de Gestió d’Ajuts Universitaris i de Recerca | Ref. 2021 SGR00693Ministerio de Ciencia, Innovación y Universidades | Ref. FPU18/00850Ministerio de Universidades | Ref. 33.50.460A.75

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