Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 µm coronatine plus 50 mm of randomly methylated-ß-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling.

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.

Enhancing Centelloside Production in Centella asiatica Hairy Root Lines through Metabolic Engineering of Triterpene Biosynthetic Pathway Early Genes.

Centella asiatica is a medicinal plant with a rich tradition of use for its therapeutic properties. Among its bioactive compounds are centellosides, a group of triterpenoid secondary metabolites whose potent pharmacological activities have attracted significant attention. Metabolic engineering has emerged as a powerful biotechnological tool to enhance the production of target compounds. In this study, we explored the effects of overexpressing the squalene synthase (SQS) gene and transcription factor TSAR2 on various aspects of C. asiatica hairy root lines: the expression level of centelloside biosynthetic genes, morphological traits, as well as squalene, phytosterol, and centelloside content. Three distinct categories of transformed lines were obtained: LS, harboring At-SQS; LT, overexpressing TSAR2; and LST, simultaneously carrying both transgenes. These lines displayed noticeable alterations in morphological traits, including changes in branching rate and biomass production. Furthermore, we observed that the expression of T-DNA genes, particularly aux2 and rolC genes, significantly modulated the expression of pivotal genes involved in centelloside biosynthesis. Notably, the LS lines boasted an elevated centelloside content but concurrently displayed reduced phytosterol content, a finding that underscores the intriguing antagonistic relationship between phytosterol and triterpene pathways. Additionally, the inverse correlation between the centelloside content and morphological growth values observed in LS lines was countered by the action of TSAR2 in the LST and LT lines. This difference could be attributed to the simultaneous increase in the phytosterol content in the TSAR2-expressing lines, as these compounds are closely linked to root development. Overall, these discoveries offer valuable information for the biotechnological application of C. asiatica hairy roots and their potential to increase centelloside production.

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.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line.

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

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 µM of coronatine (COR) or 150 µM 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.

Genetic approaches in improving biotechnological production of taxanes: An update.

Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agents in the treatment of various types of cancer. Due to the scarcity of PTX in nature, its production in cell cultures and plant organs is a major challenge for plant biotechnology. Although significant advances have been made in this field through the development of metabolic engineering and synthetic biology techniques, production levels remain insufficient to meet the current market demand for these powerful anticancer drugs. A key stumbling block is the difficulty of genetically transforming the gymnosperm Taxus spp. This review focuses on the progress made in improving taxane production through genetic engineering techniques. These include the overexpression of limiting genes in the taxane biosynthetic pathway and transcription factors involved in its regulation in Taxus spp. cell cultures and transformed roots, as well as the development and optimization of transformation techniques. Attempts to produce taxanes in heterologous organisms such as bacteria and yeasts are also described. Although promising results have been reported, the transfer of the entire PTX metabolic route has not been possible to date, and taxane biosynthesis is still restricted to Taxus cells and some endophytic fungi. The development of a synthetic organism other than Taxus cells capable of biotechnologically producing PTX will probably have to wait until the complete elucidation of its metabolic pathway.

Biodiversity, Biochemical Profiling, and Pharmaco-Commercial Applications of Withania somnifera: A Review.

Withania somnifera L. Dunal (Ashwagandha), a key medicinal plant native to India, is used globally to manage various ailments. This review focuses on the traditional uses, botany, phytochemistry, and pharmacological advances of its plant-derived constituents. It has been reported that at least 62 crucial and 48 inferior primary and secondary metabolites are present in the W. somnifera leaves, and 29 among these found in its roots and leaves are chiefly steroidal compounds, steroidal lactones, alkaloids, amino acids, etc. In addition, the whole shrub parts possess various medicinal activities such as anti-leukotriene, antineoplastic, analgesic, anti-oxidant, immunostimulatory, and rejuvenating properties, mainly observed by in vitro demonstration. However, the course of its medical use remains unknown. This review provides a comprehensive understanding of W. somnifera, which will be useful for mechanism studies and potential medical applications of W. somnifera, as well as for the development of a rational quality control system for W. somnifera as a therapeutic material in the future.

Using machine learning to link the influence of transferred Agrobacterium rhizogenes genes to the hormone profile and morphological traits in Centella asiatica hairy roots.

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.

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-ß-cyclodextrins (ß-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 ß - 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 + ß - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells.

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 metabolism 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 biosynthesized 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.

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.

The Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits Paclitaxel in vitro Biotechnological Production.

Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.

Phenylpropanoids in Silybum marianum cultures treated with cyclodextrins coated with magnetic nanoparticles.

The glucose oligosaccharide-derived cyclodextrins (CDs) are used for improving bioactive compound production in plant cell cultures because, in addition to their elicitation activity, CDs promote product removal from cells. However, despite these advantages, the industrial application of CDs is hampered by their high market price. A strategy to overcome this constraint was recently tested, in which reusable CD polymers coated with magnetic Fe3O4 nanoparticles were harnessed in Vitis vinifera cell cultures to produce t-resveratrol (t-R). In this study, we applied hydroxypropyl-ß-CDs (HPCD) and HPCDs coated with magnetic nanoparticles (HPCD-EPI-MN) in methyl jasmonate (MJ)-treated transgenic Silybum marianum cultures ectopically expressing either a stilbene synthase gene (STS) or a chalcone synthase gene (CHS), and compared their effects on the yields of t-R and naringenin (Ng), respectively. HPCD-EPI-MN at 15 g/L stimulated the accumulation of metabolites in the culture medium of the corresponding transgenic cell lines, with up to 4 mg/L of t-R and 3 mg/L of Ng released after 3 days. Similar amounts were produced in cultures treated with HPCD. Concentrations higher than 15 g/L of HPCD-EPI-MN and prolonged incubation periods negatively affected cell growth and viability in both transgenic cell lines. Reutilization of HPCD-EPI-MN was possible in three elicitation cycles (72 h each), after which the polymer retained 25-30% of its initial efficiency, indicating good stability and reusability. Due to their capacity to adsorb metabolites and their recyclability, the application of magnetic CD polymers may reduce the costs of establishing efficient secondary metabolite production systems on a commercial scale. KEY POINTS: • Long-term transgenic S. marianum suspensions stably produce transgene products • t-R and Ng accumulated extracellularly in cultures elicited with HPCD and HPCD-EPI-MN • The recyclability of HPCD-EPI-MN for metabolite production was proven.

Effect of Polyploidy Induction on Natural Metabolite Production in Medicinal Plants.

Polyploidy plays an important role in plant diversification and speciation. The ploidy level of plants is associated with morphological and biochemical characteristics, and its modification has been used as a strategy to alter the quantitative and qualitative patterns of secondary metabolite production in different medicinal plants. Polyploidization can be induced by many anti-mitotic agents, among which colchicine, oryzalin, and trifluralin are the most common. Other variables involved in the induction process include the culture media, explant types, and exposure times. Due to the effects of polyploidization on plant growth and development, chromosome doubling has been applied in plant breeding to increase the levels of target compounds and improve morphological characteristics. Prompted by the importance of herbal medicines and the increasing demand for drugs based on plant secondary metabolites, this review presents an overview of how polyploidy can be used to enhance metabolite production in medicinal plants.

Methyl-ß-cyclodextrin and coronatine as new elicitors of tropane alkaloid biosynthesis in Atropa acuminata and Atropa belladonna hairy root cultures.

Hyoscyamine (HYO) and scopolamine (SCO) are tropane alkaloids acting as anticholinergic factors on the parasympathetic nervous system in humans and are produced by Solanaceous plants. Two strains of Agrobacterium rhizogenes, A4 and LBA9402, were used to infect Atropa acuminata Royle ex Miers and Atropa belladonna L. leaf explants. A. acuminata was inoculated either by direct infection or sonicated-assisted A. rhizogenes-mediated transformation (SAAT) was performed. A. belladonna was inoculated with the A4 strain using a direct method. The selected hairy root lines of both species were elicited with 50 mM methyl-ß-cyclodextrin (ß-CD), 0.5 µM coronatine (Cor) or 50 mM ß-CD + 0.5 µM Cor on Day 14 of culture. The elicitor effect on growth and HYO and SCO content was analyzed after one (T1) and two (T2) weeks of treatment. In A. acuminata explants, the highest transformation percentage (T%) was obtained with strain A4 and the SAAT method (T%: 96.43). Cor significantly reduced the growth of A. acuminata hairy roots (fresh weight and dry weight [DW]: 2.52 and 0.3 g, respectively), whereas ß-CD increased their DW (0.4 g). Also, the combined ß-CD + Cor treatment had a positive significant effect on the DW of A. belladonna hairy roots (0.41 g). In A. acuminata hairy roots, the HYO level was lower under Cor treatment than in the control at both sampling times. In contrast, the SCO content was increased 10-fold by Cor elicitation at T1 compared to the control (10.95 mg g-1 DW) and was also positively affected by ß-CD + Cor. In A. belladonna hairy roots, all the elicitors had a negative effect on both HYO and SCO production. This report is the first assessment of the effect of ß-CD and Cor elicitors on tropane alkaloid production.

Improved biotechnological production of paclitaxel in Taxus media cell cultures by the combined action of coronatine and calix[8]arenes.

Paclitaxel (PTX), a widely used anticancer agent, is found in the inner bark of several Taxus species, although at such low levels that its extraction is ecologically unsustainable. Biotechnological platforms based on Taxus sp. cell cultures offer an eco-friendlier approach to PTX production, with yields that can be improved by elicitation. However, the also limited excretion of target compounds from the producer cells to the medium hampers their extraction and purification. In this context, we studied the effect of treating T. media cell cultures with the elicitor coronatine (COR) and calix[8]arenes (CAL), nanoparticles that can host lipophilic compounds within their macrocyclic scaffold. The highest taxane production (103.5 mg.L-1), achieved after treatment with COR (1 µM) and CAL (10 mg.L-1), was 15-fold greater than in the control, and PTX represented 82% of the total taxanes analyzed. Expression levels of the flux-limiting PTX biosynthetic genes, BAPT and DBTNBT, increased after the addition of COR, confirming its elicitor action, but not CAL. The CAL treatment significantly enhanced taxane excretion, especially when production levels were increased by COR; 98% of the total taxanes were found in the culture medium after COR + CAL treatment. By forming complexes with PTX, the nanoparticles facilitated its excretion to the medium, and by protecting cells from PTX toxicity, its intra-and extra-cellular degradation may have been avoided. The addition of COR and CAL to T. media cell cultures is therefore a bio-sustainable and economically viable system to improve the yield of this important anticancer compound.

Powerful Plant Antioxidants: A New Biosustainable Approach to the Production of Rosmarinic Acid.

Modern lifestyle factors, such as physical inactivity, obesity, smoking, and exposure to environmental pollution, induce excessive generation of free radicals and reactive oxygen species (ROS) in the body. These by-products of oxygen metabolism play a key role in the development of various human diseases such as cancer, diabetes, heart failure, brain damage, muscle problems, premature aging, eye injuries, and a weakened immune system. Synthetic and natural antioxidants, which act as free radical scavengers, are widely used in the food and beverage industries. The toxicity and carcinogenic effects of some synthetic antioxidants have generated interest in natural alternatives, especially plant-derived polyphenols (e.g., phenolic acids, flavonoids, stilbenes, tannins, coumarins, lignins, lignans, quinines, curcuminoids, chalcones, and essential oil terpenoids). This review focuses on the well-known phenolic antioxidant rosmarinic acid (RA), an ester of caffeic acid and (R)-(+)-3-(3,4-dihydroxyphenyl) lactic acid, describing its wide distribution in thirty-nine plant families and the potential productivity of plant sources. A botanical and phytochemical description is provided of a new rich source of RA, Satureja khuzistanica Jamzad (Lamiaceae). Recently reported approaches to the biotechnological production of RA are summarized, highlighting the establishment of cell suspension cultures of S. khuzistanica as an RA chemical biofactory.

Improved tropane alkaloid production and changes in gene expression in hairy root cultures of two Hyoscyamus species elicited by silicon dioxide nanoparticles.

Species of Hyoscyamus are rich sources of medicinally important tropane alkaloids, which have anticholinergic, antispasmodic and sedative effects and are competitive inhibitors of acetylcholine. The application of nanotechnology and nanomaterials for elicitation is rapidly expanding and recent research indicates that silicon dioxide nanoparticles (SiO2 NPs) can be used as an efficient elicitor to increase the production of hyoscyamine and scopolamine in Hyoscyamus species. Thus, in this work, the effect of SiO2 NPs (0, 25, 50, 100 and 200 mg L-1) with two treatment times (24 and 48 h) on the growth rate, total phenol and flavonoid content (TPC, TFC), antioxidant enzyme activity, tropane alkaloid yield and pmt (putrescine N-methyltransferase) and h6h (hyoscyamine 6-hydroxylase) gene expression levels in hairy roots of two Hyoscyamus species (H. reticulatus and H. pusillus) was investigated. The highest TPC and TFC accumulation was obtained in H. reticulatus elicited by SiO2 NPs (100 and 200 mg L-1), respectively, at 24 h of treatment. High-performance liquid chromatography (HPLC) revealed the highest amount of hyoscyamine (140.15 µg g-1 FW) and scopolamine (67.71 µg g-1 FW) accumulated in H. reticulatus transformed roots treated with 100 mg L-1 SiO2 NPs at 24 h, with a respective increase of 1212% and 272% compared to non-treated roots. In H. pusillus, the highest hyoscyamine (7.42 µg g-1 FW) and scopolamine (15.56 µg g-1 FW) production (about 82% and 241% higher, respectively, compared to the lowest amounts) was achieved with 25 and 100 mg L-1 SiO2 NPs, respectively, at 48 h of treatment. Semi-quantitative RT-PCR analysis determined the highest expression level of pmt and h6h genes in H. reticulatus transformed roots supplemented with 100 mg L-1 SiO2 NPs.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.