Search Results (14)

Taxus is the natural source of the anticancer drug paclitaxel. Although significant progress has been made in elucidating the biosynthetic pathway of paclitaxel, its tissue-specific accumulation and associated regulatory networks in Taxus remains unclear. In this study, we conducted integrated transcriptomic and metabolomic analyses of the root, leaf, shoot, bark, and wood of Taxus chinensis var. mairei to investigate the tissue-specific biosynthesis and accumulation of paclitaxel. We found that paclitaxel, 10-deacetylbaccatin III, and several taxoids were significantly enriched in the leaf, bark, and shoot, while paclitaxel derivatives, such as taxayunnansin A and taxol B, accumulated primarily in the root. Most genes involved in paclitaxel biosynthesis showed the highest expression in the root and the lowest in the wood. Using weighted gene co-expression network analysis (WGCNA), we identified several candidate transcription factors potentially regulating paclitaxel biosynthesis. Further validation using yeast one-hybrid (Y1H) and dual-luciferase reporter assays confirmed that ERF68 activates the expression of taxane-2α-hydroxylase (T2H) gene, a key gene in the paclitaxel biosynthesis pathway. Collectively, our finding provides crucial insights into the transcriptional regulation of paclitaxel biosynthesis in Taxus. Full article

BAHD acyltransferases are involved in catalyzing and regulating the secondary metabolism in plants. Despite this, the members of BAHD family and their functions have not been reported in the Taxus species. In this study, a total of 123 TwBAHD acyltransferases from Taxus wallichiana var. mairei genome were identified and divided into six clades based on phylogenetic analysis, of which Clade VI contained a Taxus-specific branch of 52 members potentially involved in taxol biosynthesis. Most TwBAHDs from the same clade shared similar conserved motifs and gene structures. Besides the typical conserved motifs within the BAHD family, the YPLAGR motif was also conserved in multiple clades of T. mairei. Moreover, only one pair of tandem duplicate genes was found on chromosome 1, with a Ka/Ks ratio < 1, indicating that the function of duplicate genes did not differentiate significantly. RNA-seq analysis revealed different expression patterns of TwBAHDs in MeJA induction and tissue-specific expression experiments. Several TwBAHD genes in the Taxus-specific branch were highly expressed in different tissues of T. mairei, suggesting an important role in the taxol pathway. This study provides comprehensive information for the TwBAHD gene family and sets up a basis for its potential functions. Full article

The presence of endophytes in plants is undeniable, but how significant their involvement is in the host plant biosynthetic pathways is still unclear. The results reported from fungicide treatments in plants varied. Fungicide treatment in Taxus was found to decrease the taxol content. In Ipomoea asarifolia, Pronto Plus and Folicur treatments coincided with the disappearance of ergot alkaloids from the plant. In Narcissus pseudonarcissus cv. Carlton, a mixture of fungicide applications decreased the alkaloids concentration and altered the carbohydrate metabolism. Jacobaea plants treated with Folicur reduced the pyrrolizidine alkaloids content. There have not been any studies into the involvement of endophytic fungi on alkaloids production of Catharanthus roseus until now. Though there is a report on the isolation of the endophytic fungi, Fusarium oxysporum from C. roseus, which was reported to produce vinblastine and vincristine in vitro. To detect possible collaborations between these two different organisms, fungicides were applied to suppress the endophytic fungi in seedlings and then measure the metabolomes by ¹HNMR and HPLC analysis. The results indicate that endophytic fungi were not directly involved in alkaloids biosynthesis. Treatment with fungicides influenced both the primary and secondary metabolism of C. roseus. The systemic fungicides Pronto Plus and Folicur caused an increase in loganin and secologanin levels. In contrast, control samples had higher level of catharanthine and vindoline. This means that fungicide treatments cause changes in plant secondary metabolism. Full article

Jasmonates (JAs) are the most effective inducers for the biosynthesis of various secondary metabolites. Currently, jasmonate ZIM domain (JAZ) and its interactors, such as MYC2, constitute the main JA signal transduction cascade, and such a cascade fails to directly regulate all the taxol biosynthesis genes, especially the rate-limit gene, DBAT. Another JA signaling branch, JAV and WRKY, would probably fill the gap. Here, TcJAV3 was the closest VQ-motif-containing protein in Taxus chinensis to AtJAV1. Although TcJAV3 was overexpressed in AtJAV1 knockdown mutant, JAVRi17, the enhanced disease resistance to Botrytis cinerea caused by silencing AtJAV1 was completely recovered. The results indicated that TcJAV3 indeed transduced JA signal as AtJAV1. Subsequently, TcWRKY26 was screened out to physically interact with TcJAV3 by using a yeast two-hybrid system. Furthermore, bimolecular fluorescence complementation and luciferase complementary imaging also confirmed that TcJAV3 and TcWRKY26 could form a protein complex in vivo. Our previous reports showed that transient TcWRKY26 overexpression could remarkably increase DBAT expression. Yeast one-hybrid and luciferase activity assays revealed that TcWRKY26 could directly bind with the w_a-box of the DBAT promoter to activate downstream reporter genes. All of these results indicated that TcWRKY26 acts as a direct regulator of DBAT, and the TcJAV3–TcWRKY26 complex is actually another JA signal transduction mode that effectively regulates taxol biosynthesis in Taxus. Our results revealed that JAV–WRKY complexes directly regulated DBAT gene in response to JA stimuli, providing a novel model for JA-regulated secondary metabolism. Moreover, JAV could also transduce JA signal and function non-redundantly with JAZ during the regulation of secondary metabolisms. Full article

Plant-associated endophytes define an important symbiotic association in nature and are established bio-reservoirs of plant-derived natural products. Endophytes colonize the internal tissues of a plant without causing any disease symptoms or apparent changes. Recently, there has been a growing interest in endophytes because of their beneficial effects on the production of novel metabolites of pharmacological significance. Studies have highlighted the socio-economic implications of endophytic fungi in agriculture, medicine, and the environment, with considerable success. Endophytic fungi-mediated biosynthesis of well-known metabolites includes taxol from Taxomyces andreanae, azadirachtin A and B from Eupenicillium parvum, vincristine from Fusarium oxysporum, and quinine from Phomopsis sp. The discovery of the billion-dollar anticancer drug taxol was a landmark in endophyte biology/research and established new paradigms for the metabolic potential of plant-associated endophytes. In addition, endophytic fungi have emerged as potential prolific producers of antimicrobials, antiseptics, and antibiotics of plant origin. Although extensively studied as a “production platform” of novel pharmacological metabolites, the molecular mechanisms of plant–endophyte dynamics remain less understood/explored for their efficient utilization in drug discovery. The emerging trends in endophytic fungi-mediated biosynthesis of novel bioactive metabolites, success stories of key pharmacological metabolites, strategies to overcome the existing challenges in endophyte biology, and future direction in endophytic fungi-based drug discovery forms the underlying theme of this article. Full article

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg²⁺, confirming the identity of Mg²⁺ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis. Full article

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-10β-hydroxylase (T10βOH) and a newly putative β-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. Full article

Fungal endophytes are well-established sources of biologically active natural compounds with many producing pharmacologically valuable specific plant-derived products. This review details typical plant-derived medicinal compounds of several classes, including alkaloids, coumarins, flavonoids, glycosides, lignans, phenylpropanoids, quinones, saponins, terpenoids, and xanthones that are produced by endophytic fungi. This review covers the studies carried out since the first report of taxol biosynthesis by endophytic Taxomyces andreanae in 1993 up to mid-2020. The article also highlights the prospects of endophyte-dependent biosynthesis of such plant-derived pharmacologically active compounds and the bottlenecks in the commercialization of this novel approach in the area of drug discovery. After recent updates in the field of ‘omics’ and ‘one strain many compounds’ (OSMAC) approach, fungal endophytes have emerged as strong unconventional source of such prized products. Full article

Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids “Taxol” is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol. Full article

Taxol is one of the potential anticancer drugs; however, the yield of Taxol and its cytotoxicity are common challenges. Thus, manipulating the Taxol biosynthetic pathway from endophytic fungi, in addition to chemical modification with biocompatible polymers, is the challenge. Four fungal isolates, namely, Aspergillus flavipes, A. terreus, A. flavus, and A. parasiticus, were selected from our previous study as potential Taxol producers, and their potency for Taxol production was evaluated in response to fluconazole and silver nitrate. A higher Taxol yield was reported in the cultures of A. flavipes (185 µg/L) and A. terreus (66 µg/L). With addition of fluconazole, the yield of Taxol was increased 1.8 and 1.2-fold for A. flavipes and A. terreus, respectively, confirming the inhibition of sterol biosynthesis and redirecting the geranyl phosphate pool to terpenoids synthesis. A significant inhibition of ergosterol biosynthesis by A. flavipes with addition of fluconazole was observed, correlating with the increase on Taxol yield. To increase the Taxol solubility and to reduce its cytotoxicity, Taxol was modified via chemical conjugation with porphyrin, and the degree of conjugation was checked from the Thin layer chromatography and UV spectral analysis. The antiproliferative activity of native and modified Taxol conjugates was evaluated; upon porphyrin conjugation, the activity of Taxol towards HepG2 was increased 1.5-fold, while its cytotoxicity to VERO cells was reduced 3-fold. Full article

Specialized plant terpenoids have found fortuitous uses in medicine due to their evolutionary and biochemical selection for biological activity in animals. However, these highly functionalized natural products are produced through complex biosynthetic pathways for which we have a complete understanding in only a few cases. Here we review some of the most effective and promising plant terpenoids that are currently used in medicine and medical research and provide updates on their biosynthesis, natural occurrence, and mechanism of action in the body. This includes pharmacologically useful plastidic terpenoids such as p-menthane monoterpenoids, cannabinoids, paclitaxel (taxol^®), and ingenol mebutate which are derived from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, as well as cytosolic terpenoids such as thapsigargin and artemisinin produced through the mevalonate (MVA) pathway. We further provide a review of the MEP and MVA precursor pathways which supply the carbon skeletons for the downstream transformations yielding these medically significant natural products. Full article

Paclitaxel is an important anticancer drug. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in Taxus, but the molecular mechanism has not yet been resolved. To establish the jasmonic acid signalling pathway of Taxus media, based on the gene of the jasmonic acid signalling pathway of Arabidopsis thaliana, sequence analysis was performed to isolate the jasmonic acid signal from the transcriptome, a transcriptional cluster of pathway gene homologs and the full length of 22 genes were obtained by RACE PCR at 5′ and 3′: two EI ubiquitin ligase genes, COI1-1 and COI1-2;7 MYC bHLH type transcription factor (MYC2, MYC3, MYC4, JAM1, JAM2, EGL3, TT8); 12 JAZ genes containing the ZIM domain; and MED25, one of the components of the transcriptional complex. The protein interaction between each were confirmed by yeast two hybridization and bimolecular fluorescence complementation based on similar genes interaction in Arabidopsis. A similar jasmonate signaling pathway was illustrated in T. media. All known paclitaxel biosynthesis genes promoters were isolated by genome walker PCR. To investigate the jasmonate signaling effect on these genes’ expression, the transcription activity of MYC2, MYC3 and MYC4 on these promoters were examined. There are 12, 10 and 11 paclitaxel biosynthesis genes promoters that could be activated by MYC2, MYC3 and MYC4. Full article

The velvet family proteins have been shown to play critical roles in fungal secondary metabolism and development. However, variations of the roles have been observed in different fungi. We report here the observation on the role of three velvet complex components VeA, VelB, and LaeA in Pestalotiopsis microspora, a formerly reported taxol-producing fungus. Deletion of individual members led to the retardation of vegetative growth and sporulation and pigmentation, suggesting critical roles in these processes. The mutant strain △velB appeared hypersensitive to osmotic stress and the dye Congo red, whereas △veA and △laeA were little affected by the pressures, suggesting only velB was required for the integrity of the cell wall. Importantly, we found that the genes played distinct roles in the biosynthesis of secondary metabolites in P. microspora. For instance, the production of pestalotiollide B, a previously characterized polyketide, required velB and laeA. In contrast, the veA gene appeared to inhibit the pestalotiollide B (PB) role in its biosynthesis. This study suggests that the three components of the velvet complex are important global regulators, but with distinct roles in hyphal growth, asexual production, and secondary metabolism in P. microspora. This work provides information for further understanding the biosynthesis of secondary metabolism in the fungus. Full article

Highly conserved, the Snf1/AMPK is a central regulator of carbon metabolism and energy production in the eukaryotes. However, its function in filamentous fungi has not been well established. In this study, we reported functional characterization of Snf1/AMPK in the growth, development and secondary metabolism in the filamentous fungus Pestalotiopsis microspora. By deletion of the yeast SNF1 homolog, we found that it regulated the utilization of carbon sources, e.g., sucrose, demonstrating a conserved function of this kinase in filamentous fungus. Importantly, several novel functions of SNF1 were unraveled. For instance, the deletion strain displayed remarkable retardation in vegetative growth and pigmentation and produced a diminished number of conidia, even in the presence of the primary carbon source glucose. Deletion of the gene caused damages in the cell wall as shown by its hypersensitivities to Calcofluor white and Congo red, suggesting a critical role of Snf1 in maintaining cell wall integrity. Furthermore, the mutant strain Δsnf1 was hypersensitive to stress, e.g., osmotic pressure (1 M sorbitol), drug G418 and heat shock, though the mechanism remains to be illustrated. Significantly, disruption of the gene altered the production of secondary metabolites. By high-performance liquid chromatography (HPLC) profiling, we found that Δsnf1 barely produced secondary metabolites, e.g., the known product pestalotiollide B. This study suggests that Snf1 is a key regulator in filamentous fungus Pestalotiopsis microspora concerting carbon metabolism and the filamentous growth, conidiation, cell wall integrity, stress tolerance and the biosynthesis of secondary metabolites. Full article