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.

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.

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.

Cationic Surfactants Based on Arginine-Phenylalanine and Arginine-Tryptophan: Synthesis, Aggregation Behavior, Antimicrobial Activity, and Biodegradation.

Cationic surfactants have great potential as drug vehicles and for use in gene therapy (cationic vesicles made from cationic surfactants can encapsulate RNA or DNA for cellular transfer). They can also be used as antimicrobial and antifungal agents to treat human infections. In an era of increasing antimicrobial resistance, the development of new biocompatible surfactants suitable for application as antimicrobial agents is of high interest. In this work, a library of amino acid-based surfactants was synthesized, characterized and tested for antimicrobial activity. The head group architecture (number and type of amino acids, density of cationic charge, ionic character) and the hydrophobic moiety (alkyl chain length and position of the hydrophobic group) were systematically modified, and the effect on the surfactant biological and aggregation behavior was studied. Thus, the pKa values, micellization process, antimicrobial efficiency and biodegradability were evaluated. The critical micelle concentration values of the surfactants depended on their hydrophobic character, but changes in the polar head as well as the position and length of the alkyl chain also significantly affected activity against some of the tested microorganisms. Moreover, biodegradability was closely related to the hydrophobic character of the surfactant and attachment of the alkyl chain to the polar head. The structure-activity relationships established here may open perspectives for the design of effective biodegradable antimicrobial materials that can overcome emerging resistance.

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.

Characterization of lipid droplets from a Taxus media cell suspension and their potential involvement in trafficking and secretion of paclitaxel.

KEY MESSAGE: Our paper describes the potential roles of lipid droplets of Taxus media cell suspension in the biosynthesis and secretion of paclitaxel and, therefore, highlights their involvement in improving its production. Paclitaxel (PTX) is a highly potent anticancer drug that is mainly produced using Taxus sp. cell suspension cultures. The main purpose of the current study is to characterize cellular LDs from T. media cell suspension with a particular focus on the biological connection of their associated proteins, the caleosins (CLOs), with the biosynthesis and secretion of PTX. A pure LD fraction obtained from T. media cells and characterized in terms of their proteome. Interestingly, the cellular LD in T. media sequester the PTX. This was confirmed in vitro, where about 96% of PTX (C0PTX,aq [mg L-1]) in the aqueous solution was partitioned into the isolated LDs. Furthermore, silencing of CLO-encoding genes in the T. media cells led to a net decrease in the number and size of LDs. This coincided with a significant reduction in expression levels of TXS, DBAT and DBTNBT, key genes in the PTX biosynthesis pathway. Subsequently, the biosynthesis of PTX was declined in cell culture. In contrast, treatment of cells with 13-hydroperoxide C18:3, a substrate of the peroxygenase activity, induced the expression of CLOs, and, therefore, the accumulation of cellular LDs in the T. media cells cultures, thus increasing the PTX secretion. The accumulation of stable LDs is critically important for effective secretion of PTX. This is modulated by the expression of caleosins, a class of LD-associated proteins with a dual role conferring the structural stability of LDs as well as regulating lipidic bioactive metabolites via their enzymatic activity, thus enhancing the biosynthesis of PTX.