Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Endophytic fungus Pseudodidymocyrtis lobariellae KL27 promotes taxol biosynthesis and accumulation in Taxus chinensis.

BACKGROUND: Taxol from Taxus species is a precious drug used for the treatment of cancer and can effectively inhibit the proliferation of cancer cells. However, the growth of Taxus plants is very slow and the content of taxol is quite low. Therefore, it is of great significance to improve the yield of taxol by modern biotechnology without destroying the wild forest resources. Endophytic fungus which symbiosis with their host plants can promote the growth and secondary metabolism of medicinal plants. RESULTS: Here, an endophytic fungus KL27 was isolated from T. chinensis, and identified as Pseudodidymocyrtis lobariellae. The fermentation broth of KL27 (KL27-FB) could significantly promote the accumulation of taxol in needles of T. chinensis, reaching 0.361 ± 0.082 mg/g·DW (dry weight) at 7 days after KL27-FB treatment, which is 3.26-fold increase as compared to the control. The RNA-seq and qRT-PCR showed that KL27-FB could significantly increase the expression of key genes involved in the upstream pathway of terpene synthesis (such as DXS and DXR) and those in the taxol biosynthesis pathway (such as GGPPS, TS, T5OH, TAT, T10OH, T14OH, T2OH, TBT, DBAT and PAM), especially at the early stage of the stimulation. Moreover, the activation of jasmonic acid (JA) biosynthesis and JA signal transduction, and its crosstalk with other hormones, such as gibberellin acid (GA), ethylene (ET) and salicylic acid (SA), explained the elevation of most of the differential expressed genes related to taxol biosynthesis pathway. Moreover, TF (transcriptional factor)-encoding genes, including MYBs, ethylene-responsive transcription factors (ERFs) and basic/helix-loop-helix (bHLH), were detected as differential expressed genes after KL27-FB treatment, further suggested that the regulation of hormone signaling on genes of taxol biosynthesis was mediated by TFs. CONCLUSIONS: Our results indicated that fermentation broth of endophytic fungus KL27-FB could effectively enhance the accumulation of taxol in T. chinensis needles by regulating the phytohormone metabolism and signal transduction and further up-regulating the expression of multiple key genes involved in taxol biosynthesis. This study provides new insight into the regulatory mechanism of how endophytic fungus promotes the production and accumulation of taxol in Taxus sp.

Taxus yunnanensis genome offers insights into gymnosperm phylogeny and taxol production.

Taxol, a natural product derived from Taxus, is one of the most effective natural anticancer drugs and the biosynthetic pathway of Taxol is the basis of heterologous bio-production. Here, we report a high-quality genome assembly and annotation of Taxus yunnanensis based on 10.7 Gb sequences assembled into 12 chromosomes with contig N50 and scaffold N50 of 2.89 Mb and 966.80 Mb, respectively. Phylogenomic analyses show that T. yunnanensis is most closely related to Sequoiadendron giganteum among the sampled taxa, with an estimated divergence time of 133.4-213.0 MYA. As with most gymnosperms, and unlike most angiosperms, there is no evidence of a recent whole-genome duplication in T. yunnanensis. Repetitive sequences, especially long terminal repeat retrotransposons, are prevalent in the T. yunnanensis genome, contributing to its large genome size. We further integrated genomic and transcriptomic data to unveil clusters of genes involved in Taxol synthesis, located on the chromosome 12, while gene families encoding hydroxylase in the Taxol pathway exhibited significant expansion. Our study contributes to the further elucidation of gymnosperm relationships and the Taxol biosynthetic pathway.

The Taxus genome provides insights into paclitaxel biosynthesis.

The ancient gymnosperm genus Taxus is the exclusive source of the anticancer drug paclitaxel, yet no reference genome sequences are available for comprehensively elucidating the paclitaxel biosynthesis pathway. We have completed a chromosome-level genome of Taxus chinensis var. mairei with a total length of 10.23 gigabases. Taxus shared an ancestral whole-genome duplication with the coniferophyte lineage and underwent distinct transposon evolution. We discovered a unique physical and functional grouping of CYP725As (cytochrome P450) in the Taxus genome for paclitaxel biosynthesis. We also identified a gene cluster for taxadiene biosynthesis, which was formed mainly by gene duplications. This study will facilitate the elucidation of paclitaxel biosynthesis and unleash the biotechnological potential of Taxus.

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway.

Taxus, commonly known as yew, is a well-known gymnosperm with great ornamental and medicinal value. In this study, by assembling a chromosome-level genome of the Himalayan yew (Taxus wallichiana) with 10.9 Gb in 12 chromosomes, we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome, resulting in the main genes for paclitaxel biosynthesis, i.e. those encoding the taxadiene synthase, P450s, and transferases, being clustered on the same chromosome. The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization. Furthermore, we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway. The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodiversity of taxoids in gymnosperms.

LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Omic analysis of the endangered Taxaceae species Pseudotaxus chienii revealed the differences in taxol biosynthesis pathway between Pseudotaxus and Taxus yunnanensis trees.

BACKGROUND: Taxol is an efficient anticancer drug accumulated in Taxus species. Pseudotaxus chienii is an important member of Taxaceae, however, the level of six taxoids in P. chienii is largely unknown. RESULTS: High accumulation of 10-DAB, taxol, and 7-E-PTX suggested that P. chienii is a good taxol-yielding species for large-scale cultivation. By the omics approaches, a total of 3,387 metabolites and 61,146 unigenes were detected and annotated. Compared with a representative Taxus tree (Taxus yunnanensis), most of the differentially accumulated metabolites and differential expressed genes were assigned into 10 primary and secondary metabolism pathways. Comparative analyses revealed the variations in the precursors and intermediate products of taxol biosynthesis between P. chienii and T. yunnanensis. Taxusin-like metabolites highly accumulated in P. chienii, suggesting a wider value of P. chienii in pharmaceutical industry. CONCLUSIONS: In our study, the occurrence of taxoids in P. chienii was determined. The differential expression of key genes involved in the taxol biosynthesis pathway is the major cause of the differential accumulation of taxoids. Moreover, identification of a number of differentially expressed transcription factors provided more candidate regulators of taxol biosynthesis. Our study may help to reveal the differences between Pseudotaxus and Taxus trees, and promote resource utilization of the endangered and rarely studied P. chienii.

Rerouting plant terpene biosynthesis enables momilactone pathway elucidation.

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

The Research Progress of Taxol in Taxus.

BACKGROUND: Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. OBJECTIVE: In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. RESULTS: The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. CONCLUSION: Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

Research progress on the source, production, and anti-cancer mechanisms of paclitaxel.

Paclitaxel, a tetracyclic diterpenoid compounds, was firstly isolated from the bark of the Pacific yew trees. Currently, as a low toxicity, high efficiency, and broad-spectrum natural anti-cancer drug, paclitaxel has been widely used against ovarian cancer, breast cancer, uterine cancer, and other cancers. As the matter of fact, natural paclitaxel from Taxus species has been proved to be environmentally unsustainable and economically unfeasible. For this reason, researchers from all over the world are devoted to searching for new ways of obtaining paclitaxel. At present, other methods, including artificial cultivation of Taxus plants, microbial fermentation, chemical synthesis, tissue and cell culture have been sought and developed subsequently. Meanwhile, the biosynthesis of paclitaxel is also an extremely attractive method. Unlike other anti-cancer drugs, paclitaxel has its unique anti-cancer mechanisms. Here, the source, production, and anti-cancer mechanisms of paclitaxel were summarized and reviewed, which can provide theoretical basis and reference for further research on the production, anti-cancer mechanisms and utilization of paclitaxel.

Evaluation of spore inoculum and confirmation of pathway genetic blueprint of T13αH and DBAT from a Taxol-producing endophytic fungus.

Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L-1 and 128.3 µg L-1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

Detection, Purification and Elucidation of Chemical Structure and Antiproliferative Activity of Taxol Produced by Penicillium chrysogenum.

Penicillium chrysogenum has been reported as a potent taxol producer based on quantitative analysis by TLC and HPLC. The biosynthetic potency of taxol has been validated from PCR detection of rate-limiting genes of taxol synthesis such as taxadienesynthase and 10-de-acetylbaccatin III-O-acetyltransferase (DBAT), which catalyzes the immediate diterpenoid precursor of the taxol substance, as detected by PCR. Taxol production by P. chrysogenum was assessed by growing the fungus on different media. Potato dextrose broth (PDB) was shown to be the best medium for obtaining the higher amount of taxol (170 µg/L). A stepwise optimization of culture conditions necessary for production of higher amounts of taxol was investigated. The substance taxol was produced optimally after 18 d of incubation at 30 °C in PDB adjusted initially at pH 8.0 with shaking (120 rpm) (250 µg/L). The P. chrysogenum taxol was purified successfully by HPLC. Instrumental analyzes such as Fourier transform infrared spectroscopy (FTIR), ultraviolet (UV) spectroscopy, 1HNMR and 13C NMR approved the structural formula of taxol (C47H51NO14), as constructed by ChemDraw. The P. chrysogenum taxol showed promising anticancer activity.

Modeling of paclitaxel biosynthesis elicitation in Corylus avellana cell culture using adaptive neuro-fuzzy inference system-genetic algorithm (ANFIS-GA) and multiple regression methods.

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-ß-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94-0.97) as compared to regression models (0.16-0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 µg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.

Whole fungal elicitors boost paclitaxel biosynthesis induction in Corylus avellana cell culture.

Paclitaxel is an effective natural-source chemotherapeutic agent commonly applied to treat a vast range of cancers. In vitro Corylus avellana culture has been reported as a promising and inexpensive system for paclitaxel production. Fungal elicitors have been made known as the most efficient strategy for the biosynthesis induction of secondary metabolites in plant in vitro culture. In this research, C. avellana cell suspension culture (CSC) was exposed to cell extract (CE) and culture filtrate (CF) derived from Camarosporomyces flavigenus, either individually or combined treatment, in mid and late log phase. There is no report on the use of whole fungal elicitors (the combined treatment of CE and CF) for the elicitation of secondary metabolite biosynthesis in plant in vitro culture. The combined treatment of CE and CF significantly led to more paclitaxel biosynthesis and secretion than the individual use of them. Also, multivariate statistical approaches including stepwise regression (SR), ordinary least squares regression (OLSR), principal component regression (PCR) and partial least squares regression (PLSR) were used to model and predict paclitaxel biosynthesis and secretion. Based on value account for (VAF), root mean square error (RMSE), coefficient of determination (R2), mean absolute percentage error (MAPE) and relative percent difference (RPD) can be concluded that mentioned regression models effectively worked only for modeling and predicting extracellular paclitaxel portion in C. avellana cell culture.

Production of the anticancer drug taxol by the endophytic fungus Epicoccum nigrum TXB502: enhanced production by gamma irradiation mutagenesis and immobilization technique.

Taxol, a phyto-extracted diterpenoid, is the most commercially needed drug in cancer chemotherapy. In spite of the microbial production of taxol being successful and prospective, the reported yields are still not sufficient for large-scale production. Thus, the discovery of new taxol-producing microbial strains and production enhancement methodologies such as process optimization, strain improvement, and immobilization technique are the main objectives. In this paper, a taxol-producing start strain Epicoccum nigrum TXB502 (initial yield 61.35 µg L-1) was isolated from Taxus baccata and identified by morphological and molecular tools. The optimum cultivation and nutritional conditions were assessed by testing one parameter at a time approach that resulted in 88.59% significant production increase. In addition, a stable mutant with improved productivity (40.07% yield increase in comparison with the parent strain) was successfully developed after gamma irradiation mutagenesis of the start strain. The taxol titer was further improved via testing different immobilization carriers for both spores and mycelia of this mutant. Over taxol production was achieved using alginate-immobilized mycelia with the feasibility of conducting six successive production cycles in a semi-continuous form. The final total concentration reached 8187.77 µg taxol 6 L-1 which represents approximately 22-fold increase, as compared to the initial titer of the start strain. These findings can pave the way for the prospective industrial manufacturing of taxol, as the achieved taxol production in this study is the highest reported by academic laboratories for microbial cultures. KEY POINTS: • Discovery of a new taxol-producing endophytic fungus E. nigrum TXB502 strain. • Taxol yield was successfully improved via bioprocess optimization and strain mutagenesis. • Alginate-immobilized mycelia were efficient for a semi-continuous production of taxol. • The final total concentration of taxol showed approximately 22-fold increase as compared to the initial titer.

Comparative transcriptome analysis of a taxol-producing endophytic fungus, Aspergillus aculeatinus Tax-6, and its mutant strain.

Taxol is a rare but extremely effective antitumor agent extracted from Taxus yew barks. Taxus plants are valuable and rare species, and the production of taxol from them is a complex process. Therefore, taxol-producing endophytic fungi seem to be a promising alternative because of their high practical value and convenient progress. In this study, the transcriptome of an endophytic fungus, Aspergillus aculeatinus Tax-6 was analyzed in order to understand the molecular mechanisms of producing fungal taxol. The results showed that genes involved in the mevalonate (MVA) pathway and non-mevalonate (MEP) pathway were expressed, including isopentenyl pyrophosphate transferase, geranyl pyrophosphate transferase, and geranylgeranyl pyrophosphate synthetase. However, those downstream genes involved in the conversion of taxa-4(5)-11(12)-diene from geranylgeranyl pyrophosphate were not expressed except for taxane 10-beta-hydroxylase. Additionally, a mutant strain, A. aculeatinus BT-2 was obtained from the original strain, A. aculeatinus Tax-6, using fungicidin as the mutagenic agent. The taxol yield of BT-2 was 560 µg L-1, which was higher than that of Tax-6. To identify the mechanism of the difference in taxol production, we compared the transcriptomes of the two fungi and explored the changes in the gene expression between them. When compared with the original strain, Tax-6, most genes related to the MVA pathway in the mutant strain BT-2 showed upregulation, including GGPPS. Moreover, most of the downstream genes were not expressed in the mutant fungi as well. Overall, the results revealed the pathway and mechanism of taxol synthesis in endophytic fungi and the potential for the construction of taxol-producing genetic engineering strains.