Taxol Production Research Articles

Callus induction, subculture, and browning inhibition of the anticancer plant Taxus media

In order to obtain more effective cell culture parameters of Taxus anticancer plants, we optimized the callus induction and subculture conditions of the explants (stem segments with buds) of the anticancer medicinal plant Taxus media by using the plant tissue culture technology and orthogonal test. Furthermore, we studied the method to inhibit browning in the culture. The results indicated that the optimal conditions for inducing callus was culture in the medium composed of B5+0.25 mg/L 2, 4-D+1.5 mg/L NAA+0.5 mg/L KT in the dark, which showed short induction time and a high induction rate (86.7%). The formula of the optimal medium for subculture was B5+0.5 mg/L 2, 4-D+2.0 mg/L NAA+1.5 mg/L KT.The proliferation multiple of callus cultured by subculture on the 10th day of callus growth was the highest. Activated carbon inhibited the browning in callus subculture, with the optimal inhibitory concentration of 0.8 g/L. The results of this study lay a foundation for the production of taxol by suspension culture of T. media cells.

Fungal endophytes of Taxus species and regulatory effect of two strains on taxol synthesis

BackgroundTaxol, derived from Taxus trees, is a valuable natural resource for the development of anticancer drugs. Endophytic fungi from Taxus trees are a promising alternative source of Taxol. However, the impact of plant-endophytic microbial interaction on the host’s Taxol biosynthesis is largely unknown.ResultsIn the current study, the diversity of endophytic fungi in three different Taxus species was analyzed using Internal Transcribed Spacer sequencing. A total of 271 Operational Taxonomic Units (OTUs) were identified, grouping into 2 phyla, 8 classes, 16 orders, 19 families, and 19 genera. Alpha and beta diversity analysis indicated significant differences in endophytic fungal communities among the various Taxus trees. At the genus level, Alternaria and Davidiella were predominantly found in T. mairei and T. media, respectively. By utilizing a previously published dataset, a Pearson correlation analysis was conducted to predict the taxol biosynthesis-related fungal genera. Following screening, two isolates of Alternaria (L7 and M14) were obtained. Effect of inoculation with Alternaria isolates on the gene expression and metabolite accumulation of T. mairei was determined by transcriptomic and untargeted metabolomic studies. The co-inoculation assay suggests that the two Alternaria isolates may have a negative regulatory effect on taxol biosynthesis by influencing hormone signaling pathways.ConclusionOur findings will serve as a foundation for advancing the production and utilization of Taxus and will also aid in screening endophytic fungi related to taxol production.

Corylus avellana as a potential alternative source for the production of taxol and taxanes

This paper demonstrates the increased interest that scientists have recently shown in the non-food components of the hazel plant, such as leaves, as well as hard and green shells, in addition to the nuts. The review discusses studies conducted to determine the qualitative and quantitative profiles of different parts of Corylus avellana. It has been demonstrated that extracts made from various Corylus avellana components possess a variety of biological properties, such as anticancer, antiproliferative, and antioxidant effects. It has been shown that secondary metabolites can be directly synthesized by in vitro cell culture, which allows the production of valuable secondary metabolites such as paclitaxel, one of the most important anticancer compounds synthesized by hazel. A brief review of existing literature data serves to emphasize C.avellana as a source of bioactive compounds. Further research is needed to explore the health benefits of C.avellana extracts and their use as food supplements. Keywords: Hazel, Corylus avellana, secondary metabolites, taxol, anticancer drug

Production and bioprocessing of Taxol from Aspergillus niger, an endophyte of Encephalartos whitelockii, with a plausible biosynthetic stability: antiproliferative activity and cell cycle analysis

The biosynthetic potency of Taxol by fungi raises their prospective to be a platform for commercial production of Taxol, nevertheless, the attenuation of its productivity with the fungal storage, is the challenge. Thus, screening for a novel fungal isolate inhabiting ethnopharmacological plants, with a plausible metabolic stability for Taxol production could be one of the most affordable approaches. Aspergillus niger OR414905.1, an endophyte of Encephalartos whitelockii, had the highest Taxol productivity (173.9 μg/L). The chemical identity of the purified Taxol was confirmed by HPLC, FTIR, and LC–MS/MS analyses, exhibiting the same molecular mass (854.5 m/z) and molecular fragmentation pattern of the authentic Taxol. The purified Taxol exhibited a potent antiproliferative activity against HepG-2, MCF-7 and Caco-2, with IC50 values 0.011, 0.016, and 0.067 μM, respectively, in addition to a significant activity against A. flavus, as a model of human fungal pathogen. The purified Taxol displayed a significant effect against the cellular migration of HepG-2 and MCF-7 cells, by ~ 52–59% after 72 h, compared to the control, confirming its interference with the cellular matrix formation. Furthermore, the purified Taxol exhibited a significant ability to prompt apoptosis in MCF-7 cells, by about 11-fold compared to control cells, suppressing their division at G2/M phase. Taxol productivity by A. niger has been optimized by the response surface methodology with Plackett–Burman Design and Central Composite Design, resulting in a remarkable ~ 1.6-fold increase (279.8 μg/L), over the control. The biological half-life time of Taxol productivity by A. niger was ~ 6 months of preservation at 4 ℃, however, the Taxol yield by A. niger was partially restored in response to ethyl acetate extracts of E. whitelockii, ensuring the presence of plant-derived signals that triggers the cryptic Taxol encoding genes.

Insights into taxadiene synthase catalysis and promiscuity facilitated by mutability landscape and molecular dynamics

Main conclusionProtein modeling, carbocation docking, and molecular dynamics along with structure-based mutability landscapes provided insight into taxadiene synthase catalysis (first step of the anticancer Taxol biosynthesis), protein structure–function correlations, and promiscuity.Plant terpenes belong to one of the largest and most diverse classes of natural products. This diversity is driven by the terpene synthase enzyme family which comprises numerous different synthases, several of which are promiscuous. Taxadiene synthase (TXS) is a class I diterpene synthase that catalyzes the first step in the biosynthesis pathway of the diterpene Taxol, an anticancer natural product produced by the Taxus plant. Exploring the molecular basis of TXS catalysis and its promiscuous potential garnered interest as a necessary means for understanding enzyme evolution and engineering possibilities to improve Taxol biosynthesis. A catalytically active closed conformation TXS model was designed using the artificial intelligence system, AlphaFold, accompanied by docking and molecular dynamics simulations. In addition, a mutability landscape of TXS including 14 residues was created to probe for structure–function relations. The mutability landscape revealed no mutants with improved catalytic activity compared to wild-type TXS. However, mutations of residues V584, Q609, V610, and Y688 showed high degree of promiscuity producing cembranoid-type and/or verticillene-type major products instead of taxanes. Mechanistic insights into V610F, V584M, Q609A, and Y688C mutants compared to the wild type revealed the trigger(s) for product profile change. Several mutants spanning residues V584, Q609, Y688, Y762, Q770, and F834 increased production of taxa-4(20),11(12)-diene which is a more favorable substrate for Taxol production compared to taxa-4(5),11(12)-diene. Finally, molecular dynamics simulations of the TXS reaction cascade revealed residues involved in ionization, carbocation stabilization, and cyclization ushering deeper understanding of the enzyme catalysis mechanism.Graphical abstract

Evaluation of phytochemical responses and paclitaxel content of yew (Taxus baccata) seedlings subjected to the systemic fungicides

AbstractEndophytic fungi pass all or at least a part of their life cycle inside or between host cells in living plant tissues. These fungi are not capable of causing disease in the host plants, however, they are deemed to be associated with the production of some secondary metabolites in plants. Therefore, this study was laid out to shed light on how fungicides are involved in the formation of key metabolites of yew (Taxus baccata L.), especially taxol. Through the symbiotic fungal endophyte activities and population changes an experiment was conducted as a completely randomized block design using two foliar application treatments of Rovral‐TS and Fosetyl aluminium fungicides. Plants sprayed with distilled water were used as a control. The findings revealed that the diversity of endophytic fungi declined with the application of systemic fungicides, leading to a decrease in the production of certain secondary metabolites. Specifically, the levels of paclitaxel decreased by 35%, while the photosynthetic pigments chlorophyll‐a, chlorophyll‐b and total chlorophyll decreased by 26%, 20.6% and 19.6%, respectively. Additionally, the levels of carotenoids, total phenol and total flavonoids decreased by 18%, 15% and 21.8%, respectively. The highest amount of these compounds was observed in the control treatment. An increase in antioxidant activity, soluble sugars and proline content was observed soon after fungicides application. The variation in the content of soluble sugars, proline and antioxidant activity was fungicide‐dependent. Compared to Fosetyl aluminium fungicide, the numerical value of the above parameters in plants treated with Rovral‐TS was 14.4%, 11.7% and 25.4% higher, respectively. The fungicide‐mediated induction of Taxol production—key secondary metabolites in yew—was to a great extent associated with the change in endophytic fungi. This further establishes the role of fungal endophytes in the stimulation of secondary metabolite formation in yew plants.

Synthesis Method of Taxol And Its Application in Cancer Treatment

Taxol, also known as paclitaxel, it is a broad known anticancer compound. It is a medication that can be used to treat multiple cancer. Taxol comes mainly from the pacific yew tree bark and it is the first microtube agent. It has been approved that taxol is a significantly chemotherapeutic agent against many forms of cancer, it has been widely used on patients. Unfortunately, it has limited production amount, chemical and biological synthesis have been worldwide researched and tested to improve taxol production. Following the background and methodology of synthesis of taxol, the different methods and procedures investigated will have a brief explanation of some of the known processes. Also, summarization of paclitaxel anticancer mechanism, and immunomodulatory effects explained here. Taxol can be used to treat breast cancer as a first-line therapy. Here, the discussion centered around current research that focus on taxol treatment for breast cancer, its mechanisms of treating cancer and effects on breast cancer.

Improved production of Taxol® precursors in S. cerevisiae using combinatorial in silico design and metabolic engineering

BackgroundIntegrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells.ResultsUsing constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae.ConclusionsThis study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.

Overcoming seed dormancy of Taxus baccata L. by embryo rescue leads to germination and seedling growth

Hairy roots generated by Rhizobium rhizogenes-mediated transformation of yew (Taxus spp.) is a promising approach to enhance production of Taxol® (paclitaxel), which is one of the most effective anticancer drugs. As a prerequisite, it is pivotal to successfully produce Taxus seedlings for transformation. However, the deep dormancy of Taxus seeds leads to extreme difficulties in seed germination. Therefore, embryo rescue has been used to break the dormancy of Taxus baccata seeds, thereby producing seedlings for transformation. In the current study, a successful strategy of embryo rescue was to sterilize the surface of T. baccata seeds collected from the field at two different maturity stages (low and high). The strategy resulted in 100% germination rate, but it was worth noting that not all germinated embryos grew into fully developed plants. As a result, the present experiment introduced an innovative indicator—fully developed seedling index—to describe the growth of seedlings developed by germinated embryos. Collectively, the data revealed that 21 ± 4% of the seedlings eventually grew into fully developed plants. Regarding the development of the seedlings, the fully developed seedling index increased initially along with seedling growth, reaching a peak after 2 weeks. Subsequently, the fully developed seedling index from low maturity seeds and high maturity seeds began to decrease until it stabilized after 4 weeks and 7 weeks, respectively. Consequently, the new findings proved helpful to select T. baccata seeds with appropriate maturity, hence developing a reliable technique to produce viable seedlings for a transformation pipeline.

Alternaria alternata F3, a Novel Taxol-Producing Endophytic Fungus Isolated from the Fruits of Taxus cuspidata: Isolation, Characterization, Taxol Yield Improvement, and Antitumor Activity.

In this study, a novel taxol-producing endophytic fungus, strain F3, was isolated from the fruits of Taxus cuspidata and identified as Alternaria alternata according to its macroscopic and microscopic traits and sequence analysis of internal transcribed spacer (ITS). The presence of taxol was detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and confirmed by ultra-high-performance liquid chromatography-electrospray coupled to tandem mass spectrometry (UPLC-ESI-MS/MS) and nuclear magnetic resonance (NMR). The fermentation parameters of strain F3 were then optimized for high taxol production. The maximum taxol yield of 195.4µg L-1 by A. alternata F3 was observed in 200-mL yeast peptone dextrose (YPD) broth, at an initial pH value of 6.0, supplemented with 0.1g L-1 sodium acetate, 0.25g L-1 salicylic acid, and 0.00125g L-1 silver nitrate and inoculum size 2%, and incubated at 28°C and 150rpm for 8days, which was 2.12-fold compared with the initial yield of taxol. Also, fungal taxol exhibited antitumor activity towards human lung carcinoma (A549) cell line and human cervical carcinoma (Hela) cell line with IC50 values of 3.98µgmL-1 and 0.35µgmL-1. Overall, this is the first report on taxol-producing endophytic fungus isolated from the fruits of Taxus. This study offers a novel source for the production of taxol for anticancer treatment.

Diversity of Endophytic Microbes in Taxus yunnanensis and Their Potential for Plant Growth Promotion and Taxane Accumulation.

Taxus spp. are ancient tree species that have survived from the Quaternary glacier period, and their metabolites, such as taxol, have been used as anticancer drugs globally. Plant-endophytic microbial interaction plays a crucial role in exerting a profound impact on host growth and secondary metabolite synthesis. In this study, high-throughput sequencing was employed to explore endophytic microbial diversity in the roots, stems, and leaves of the Taxus yunnanensis (T. yunnanensis). The analysis revealed some dominant genera of endophytic bacteria, such as Pseudomonas, Neorhizobium, Acidovorax, and Flavobacterium, with Cladosporium, Phyllosticta, Fusarium, and Codinaeopsis as prominent endophytic fungi genera. We isolated 108 endophytic bacteria and 27 endophytic fungi from roots, stems, and leaves. In vitro assays were utilized to screen for endophytic bacteria with growth-promoting capabilities, including IAA production, cellulase, siderophore production, protease and ACC deaminase activity, inorganic phosphate solubilization, and nitrogen fixation. Three promising strains, Kocuria sp. TRI2-1, Micromonospora sp. TSI4-1, and Sphingomonas sp. MG-2, were selected based on their superior growth-promotion characteristics. These strains exhibited preferable plant growth promotion when applied to Arabidopsis thaliana growth. Fermentation broths of these three strains were also found to significantly promote the accumulation of taxanes in T. yunnanensis stem cells, among which strain TSI4-1 demonstrated outstanding increase potentials, with an effective induction of taxol, baccatin III, and 10-DAB contents. After six days of treatment, the contents of these metabolites were 3.28 times, 2.23 times, and 2.17 times the initial amounts, reaching 8720, 331, and 371 ng/g of dry weight of stem cells, respectively. These findings present new insight into the industrialization of taxol production through Taxus stem cell fermentation, thereby promoting the conservation of wild Taxus resources by maximizing their potential economic benefits.

Secondary Metabolism in Taxus spp. Plant Cell Culture In Vitro

The genus Taxus (yew) is a source of a number of high-value medicinal substances, particularly, paclitaxel (taxol)—a complex diterpenoid compound with a powerful antitumor action (trade name of Taxol®). Paclitaxel is one of the most efficient drugs in chemotherapy owing to its specific ability to suppress proliferation of tumor cells via stabilization of their microtubules. The world-wide demand for taxol is 800–1000 kg a year and these figures annually rise by 20%. The growing need for paclitaxel and its derivatives and the shortage of plant resources necessary for their production made compounds of the taxane group one of the most important objects for development of biotechnological methods of their production. Out of all the possible ways of taxol production (isolation from wild or plantation trees, total chemical synthesis or semisynthesis, use of yew cell cultures, techniques of metabolic engineering, and use of yew endophytic fungi), the most promising is industrial cultivation of Taxus spp. cell cultures. This review examines the papers dealing with investigation of secondary metabolism in dedifferentiated cells in vitro of various yew species and feasibility of industrial use of cell cultures for production of taxoids. We revealed a number of specificity of Taxus spp. cell cultures: (1) from a cytophysiological aspect—difficult initiation of cell cultures, their low growth characteristics, specific media and culturing conditions; (2) from a phytochemical aspect—distinction from intact plants in qualitative composition and content of secondary metabolites accounted for by specificity of cell culture as a biological system; predominant formation of С14-hydroxylated rather than of С13-hydroxylated taxoids; an opportunity for elevation of the content of taxoids—including commercially valuable ones (paclitaxel and baccatin III) with the aid of different tools (elicitation, stress exposures, two-phase cultivation and some others); (3) from a biotechnological aspect—possibility of industrial cultivation of yew cell cultures; existence of several successful industries (Germany and the Republic of Korea).

Secondary metabolism in <i>Taxus</i> spp. plant cell culture in vitro

The genus Taxus (yew) is a source of a number of high-value medicinal substances, particularly, paclitaxel (taxol)a complex diterpenoid compound with a powerful antitumor action (trade name of Taxol). Paclitaxel is one of the most efficient drugs in chemotherapy owing to its specific ability to suppress proliferation of tumor cells via stabilization of their microtubules. The world-wide demand for taxol is 8001000 kg a year and these figures annually rise by 20%. The growing need for paclitaxel and its derivatives and the shortage of plant resources necessary for their production made compounds of the taxane group one of the most important objects for development of biotechnological methods of their production. Out of all the possible ways of taxol production (isolation from wild or plantation trees, total chemical synthesis or semisynthesis, use of yew cell cultures, techniques of metabolic engineering, and use of yew endophytic fungi), the most promising is industrial cultivation of Taxus spp. cell cultures. This review examines the papers dealing with investigation of secondary metabolism in dedifferentiated cells in vitro of various yew species and feasibility of industrial use of cell cultures for production of taxoids. We revealed a number of specificity of Taxus spp. Cell cultures: (1) from a cytophysiological aspectdifficult initiation of cell cultures, their low growth characteristics, specific media and culturing conditions; (2) from a phytochemical aspectdistinction from intact plants in qualitative composition and content of secondary metabolites accounted for by specificity of cell culture as a biological system; predominant formation of С14-hydroxylated rather than of С13-hydroxylated taxoids; an opportunity for elevation of the content of taxoidsincluding commercially valuable ones (paclitaxel and baccatin III) with the aid of different tools (elicitation, stress exposures, two-phase cultivation and some others); (3) from a biotechnological aspectpossibility of industrial cultivation of yew cell cultures; existence of several successful industries (Germany and the Republic of Korea).

The Stimulation of the Anticancer Chemical Taxol in Taxus baccata

Abstract Taxol is an anticancer drug that is widely used in cancer treatment worldwide. Since the source of the drug is a plant, the increase in its production is one of the concerns of researchers. One way to increase the production of Taxol is to use stimulants for secondary metabolite production. Therefore, in this study, methyl jasmonate was used as a bioelicitor, and its effect on Taxol production was evaluated. For this purpose, Taxus baccata was elicited for 48 and 72 hours with concentrations at 0, 100, 250, and 500 μM methyl jasmonate, and the quantities of Taxol in the leaf and stem explants were measured by high-performance liquid chromatography (HPLC). The results showed that among the leaf and stem explants, a tremendous amount of Taxol production is related to the leaf, which is a great advantage in reducing the number of trees cut down to obtain Taxol. In addition, the 48-hour elicitation time demonstrated the best result in the production of Taxol. The optimal concentration of methyl jasmonate was also estimated at 500 μM and 0.326 mg/g of dried leaf biomass. The results of this experiment showed that methyl jasmonate has the potential to be used as a biological elicitor to produce considerable amounts of Taxol in Taxus baccata.

Production of Taxol by Endophytic Fungi Isolated from Roots of Himalayan Yew (Taxus wallichiana Zucc.)

Taxol® (generic name – Paclitaxel), the most promising chemotherapeuticagent was isolated from bark of different Taxus sp. As Taxus species arethreatened with extinction (endangered in Himalaya), thus it is imperativeto develop alternate and sustainable method for commercialization and scaleup production of paclitaxel. In this respect, physical and chemical parametersare effective and important key points for active compound production par-ticularly by using endophytic microbes. In the present study, five endophyticfungi isolated from the roots of Taxus wallichiana, are tested for paclitaxelproduction using biochemical and molecular methods. Subsequently, effectof physico-chemical parameters like temperature, pH, incubation time, andmedium constituents i.e., salt concentration, carbon and nitrogen sources onpaclitaxel production were also analyzed. Among isolates, two of the fungiviz. GBPI TWR F1 (Penicillium sp.) and GBPI TWR F5 (Aspergillus sp.)were found to be paclitaxel producing. The genomic DNA samples were sequenced to confirm the presence of two genes viz. 10-deacetylbaccatinIII-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT), implicated in paclitaxel biosynthesis. Boththe endophytes showed the amplicons of DBAT and BAPT genes. Resultsrevealed that after optimization of medium components and physical condi-tion, paclitaxel production was increased in both the endophytes, maximumpaclitaxel production i.e., 5.45 ± 0.98 mg/L was obtained by GBPI TWR F5(Aspergillus sp.) following 10 days of incubation at 15◦C in optimized S7liquid medium composition

An Overview on Taxol Production Technology and Its Applications as Anticancer Agent

An Overview on Taxol Production Technology and Its Applications as Anticancer Agent

Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

BackgroundCYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae.ResultsInvestigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses.ConclusionsThis study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.

Factors affecting the growth, antioxidant potential, and secondary metabolites production in hazel callus cultures

Hazelnut is one of the most important nut plants recently suggested as a sustainable source for paclitaxel. In the present study, the effect of the concentration and combination of PGRs, different basal medium and ultrasonic waves on callus induction and growth, physiological characteristics, and taxol and baccatin III production in hazelnut callus cultures were investigated. The results indicated that combining 2,4-D (2 mg/L) and Kin (0.2 mg/L) with the sonication of explants for 1 min provides an optimized condition for callus induction and growth. Hazelnut explants exhibited different callus production and biochemical and metabolic characteristics depending on the basal medium type, ultrasound treatment, and inclusion of ascorbic acid in the medium. So that, the highest percentage of callogenesis (100%) observed in ½ MS + 1 min US, ½ MS + 150 mg/L AA, B5 + 1 min US and B5 + 150 mg/L AA, and also ½ MS salt + Nitsch vitamins + 150 mg/L AA. Furthermore, the highest callus growth (7.86 g FW) was obtained from ½ MS + 1 min US. The highest amount of baccatin III production (147.98 and 147.85 mg/L) was obtained from the WPM and MS basal media; the highest taxol production (44.89 mg/L) was observed in the WPM basal medium. The cultures in the MS, WPM, and MS salts + Nitsch vitamins media, had the highest H2O2 and MDA content, antioxidant enzymes activity, and phenolic compounds. In conclusion, culture media nutrient composition and concentration not only affect the cell growth and physiological status of the cultures but also improve secondary metabolites production and accumulation.

Taxoid profile in endophytic fungi isolated from Corylus avellana, introduces potential source for the production of Taxol in semi-synthetic approaches

Taxol (Paclitaxel) and its derivative taxanes are widely used in chemotherapy and treatment of different types of cancer. Although the extracted taxanes from Taxus sp. are currently used in semi-synthetic production of Taxol, providing alternative always available sources is still a main concern. Due to availability and fast growth rate, microorganisms are much potent alternative sources for taxanes. In the present study, 249 endophytic fungi were isolated from Corylus avellana at six different locations of Iran, among which 18 species were capable to produce taxanes. Genotyping analysis indicated that 17 genera were ascomycetes but only one basidiomycete. Seven taxanes were detected and quantified in solid and suspension cultures by HPLC and their structures were confirmed by LC-Mass analysis. Among endophytes, CA7 had all 7 taxoids and CA1 had the highest Taxol yield. In 78% of endophytes transferring to liquid media was accompanied by increase of taxanes yield and increased taxan production and its release to media up to 90%. Evaluation of cytotoxicity indicated that extracts of all isolated fungi were lethal to MCF7 cells. Since endophytes produced remarkable amounts of taxanes, they can be suggested as alternative inexpensive and easily available resources for Taxol production in semi-synthesis plans.

Boosting the Anticancer Activity of Aspergillus flavus “endophyte of Jojoba” Taxol via Conjugation with Gold Nanoparticles Mediated by γ-Irradiation

Taxol production by fungi is one of the promising alternative approaches, regarding to the natural and semisynthetic sources; however, the lower yield and rapid loss of Taxol productivity by fungi are the major challenges that halt their further industrial implementation. Thus, searching for fungal isolates with affordable Taxol-production stability, in addition to enhance its anticancer activity via conjugation with gold nanoparticles, is the main objectives of this study. Twenty-four endophytic fungal isolates were recovered from the barks, twigs, and leaves of jojoba plant, among these fungi, Aspergillus flavus MW485934.1 was the most potent Taxol producer (88.6 µg/l). The chemical identity of the extracted Taxol of A. flavus was verified by the TLC, HPLC, HNMR, and FTIR analyses. The yield of Taxol produced by A. flavus was optimized by the response surface methodology (RSM) using Plackett–Burman (PBD) and faced central composite designs (FCCD). The yield of Taxol by A. flavus was increased by about 3.2 folds comparing to the control cultures (from 96.5 into 302.7 µg/l). The highest Taxol yield by was obtained growing A. flavus on a modified malt extract medium (g/l) (malt extract 20.0, peptone 2.0, sucrose 20.0, soytone 2.0, cysteine 0.5, glutamine 0.5, and beef extract 1.0 adjusted to pH 6.0) and incubated at 30 °C for 16 days. From the FCCD design, the significant variables affecting Taxol production by A. flavus were cysteine, pH, and incubation time. Upon A. flavus γ-irradiation at 1.0 kGy, the Taxol yield was increased by about 1.25 fold (375.9 µg/l). To boost its anticancer activity, the purified Taxol was conjugated with gold nanoparticles (AuNPs) mediated by γ-rays irradiation (0.5 kGy), and the physicochemical properties of Taxol-AuNPs composite were evaluated by UV–Vis, DLS, XRD, and TEM analyses. The IC50 values of the native-Taxol and Taxol-AuNPs conjugates towards HEPG-2 cells were 4.06 and 2.1 µg/ml, while the IC50 values against MCF-7 were 6.07 and 3.3 µg/ml, respectively. Thus, the anticancer activity of Taxol-AuNPs composite was increased by 2 folds comparing to the native Taxol towards HEPG-2 and MCF-7 cell lines. Also, the antimicrobial activity of Taxol against the multidrug resistant bacteria was dramatically increased upon conjugation with AuNPs comparing to authentic AuNPs and Taxol, ensuring the higher solubility, targetability, and efficiency of Taxol upon AuNPs conjugation.