Advances and prospects of taxol biosynthesis by endophytic fungi

BackgroundTaxol 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.ResultsHere, 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.ConclusionsOur 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 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. 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. 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. 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.

Different endophytic fungi isolated from Himalayan Yew plants were tested for their ability to produce taxol. The BAPT gene (C-13 phenylpropanoid side chain-CoA acetyl transferase) involved in the taxol biosynthetic pathway was used as a molecular marker to screen taxol-producing endophytic fungi. Taxol extracted from fungal strain TBPJ-B was identified by HPLC and MS analysis. Strain TBPJ-B was identified as Fusarium redolens based on the morphology and internal transcribed spacer region of nrDNA analysis. HPLC quantification of fungal taxol showed that F. redolens was capable of producing 66 μg/l of taxol in fermentation broth. The antitumour activity of the fungal taxol was tested by potato disc tumor induction assay using Agrobacterium tumefaciens as the tumor induction agent. The present study results showed that PCR amplification of genes involved in taxol biosynthesis is an efficient and reliable method for prescreening taxol-producing fungi. We are reporting for the first time the production of taxol by F. redolens from Taxus baccata L. subsp. wallichiana (Zucc.) Pilger. This study offers important information and a new source for the production of the important anticancer drug taxol by endophytic fungus fermentation.

More than almost any other commercially useful plant secondary metabolite, Taxol has presented a formidable challenge to the efforts to devise an economically viable and sustainable process for its commercial production. The complexity of its structure does not bode well for success of the extensive efforts to develop a commercially feasible chemical total synthesis. Thus, at least for some time to come, the industrial production of Taxol will have to rely on biosynthesis of at least the complex diterpene moiety. As detailed in chapter 5 of this text, a medium-range solution of the problem of production of Taxol and analogues has been developed based on semisynthesis from 10-deacetylbaccatin-in which can be extracted from a renewable resource, the needles of European and Himalayan yew. However, in the long run it would be highly desirable to be able to produce Taxol and analogues thereof by a process which does not have to rely on the extraction of plant material but can rather be carried out under entirely controllable conditions. Such a biotechnological process may be based on a plant cell culture fermentation, a microbial fermentation with a genetically engineered organism, or it may be a hybrid process combining either of these two approaches with chemical steps and/or the use of isolated enzymes. The development of such a process would be based on, or would at least greatly benefit from, detailed knowledge of the biosynthetic pathway by which the plant assembles Taxol and by characterization of the enzymes catalyzing the various reactions involved and of the genes coding for them. Although the few biotechnological processes for the commercial production of plant secondary metabolites currently available were all developed empirically, it seems likely that in the future genetic engineering of metabolic pathways based on an understanding of the biosynthesis will play an increasingly prominent role. It is in this context that the biosynthesis of Taxol is of interest as a starting point for the future development of alternative biotechnological production processes for Taxol and the next generations of Taxol analogues.

Fungal Taxol acquired lots of attention in the last few decades mainly because of the hope that fungi could be manipulated more easily than yew trees to scale up the production level of this valuable anticancer drug. Several researchers have studied diverse factors to enhance fungal Taxol production. However, up to date fungal Taxol production has never been enhanced to the commercial level. We have hypothesized that optimization of fungal Taxol production may require clear understanding of the fungal habitat in its original host plant. One major feature shared by all fungal endophytes is that they are located in the internal plant tissues where darkness is prominent; hence here the effect of light on fungal Taxol production was tested. Incubation of Taxol-producing endophytic SSM001 fungus in light prior to inoculation in Taxol production culture media showed dramatic loss of Taxol accumulation, significant reduction in Taxol-containing resin bodies and reduction in the expression of genes known to be involved in Taxol biosynthesis. The loss of Taxol production was accompanied by production of dark green pigments. Pigmentation is a fungal protection mechanism which is photoreceptor mediated and induced by light. Opsin, a known photoreceptor involved in light perception and pigment production, was identified in SSM001 by genome sequencing. SSM001 opsin gene expression was induced by white light. The results from this study indicated that the endophytic fungus SSM001 required the dark habitat of its host plant for Taxol production and hence this biosynthetic pathway shows a negative response to light.

Taxol is a valuable drug in the treatment of many cancers, which is extracted from plant sources, especially the Taxus plant. The restriction of plant resources has led to new solutions for increasing plant production of taxol. One of these solutions is the use of elicitors such as methyl jasmonate. In the present study, the effect of the elicitation by methyl jasmonate on the expression of genes involved in taxol biosynthesis was evaluated. For this purpose, taxus leaf and stem explants of Taxus baccata were elicited with concentrations of 0, 100, 250, and 500 μM methyl jasmonate for 48 and 72 hours, and then the expression of DBAT, BAPT, and TS genes in the different explants, concentrations, and treatment times were investigated using Real-time PCR method. The findings showed that the expression of the three genes under the elicitation of methyl jasmonate increased in the leaves and stems of the taxus plant, and their expression was higher in the leaves than in the stems. The effect of elicitation time was shown that increasing the elicitation time from 48 to 72 hours increases the expression of the three genes. Furthermore, the largest increase in expression for all three genes was observed at 250 μM methyl jasmonate. The results showed that methyl jasmonate can increase the production of taxol in Taxus baccata by increasing the expression of some genes involved in the taxol biosynthesis pathway.

One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of L-phenylalanine to trans-cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40-50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor alpha-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 microM but having no effect at 10 microM. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.

青檀叶片内生和附生真菌组成及生态分布

Nitric oxide mediates the fungal elicitor-induced taxol biosynthesis of Taxus chinensis suspension cells through the reactive oxygen species-dependent and -independent signal pathways

Endophytic fungi, microfungi that internally infect living plant tissues, are reported to have the ability to synthesize many enzymes, plant growth hormones and pharmaceutically active compounds similar to those in their hosted plants. This has opened a potential path of using endophytic fungi as a bioreactor for mass production of bioactive compounds at a lower cost. Therefore, it is necessary to establish a robust procedure for the isolation and identification of potential fungal strains that are capable of producing desired biological compounds. In this study, we reported an effective procedure for surface sterilization of 3 types of tissue samples (root, shoot and leaf) of 2 herbaceous plants (Catharanthus roseus and Scutallaria barbata) using commercial bleach (5% NaOCl), isolation of endophytic fungi from the sterilized samples and identification of isolated fungal strains by ITS sequencing analysis. A total of 48 endophytic fungi were successfully isolated from plant samples collected from Dan Phuong (Ha Noi), Phu Dien (Ha Noi) and Hai Duong city (Hai Duong). Based on results of morphological observation and ITS sequencing analysis, 48 endophytic fungi were classified to one of the four species, including Clasdosporium colombiae, Cladosporium halotolerans, Corynespora cassiicola and Albifimbria terrestris. The potential of the isolated endophytic fungal species for the mass production of pharmacologically active compounds will be investigated in future studies.

Medicinal plants such as porang (Amorphophallus muelleri Blume) can produce bioactive compounds from plants-associated endophytes. Therefore, medicinal plants were a sources of isolation of endophytic fungi and endophytic fungi were a sources of secondary metabolites that have anticancer, antimalarial, antimicrobial, and so on. A Porang tuber has been used as a medicine for boils, medicine for sliced wounds and medicine for wounds due to venomous animal bites. The research was aimed to carry out isolation and characterization of endophytic fungi from porang tuber, and to test the ability of endophytic fungi from porang tuber as antibacterial against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria. Isolation of endophytic fungi from porang tubers succeeded in getting five different fungi isolates. Based on macroscopic and microscopic characteristics, endophytic fungi isolated from porang tuber were members of the genus Curvularia, Penicillium, 2 isolates of Aspergillus, and 1 isolate that had not been identified. Porang endophytic fungi had antibacterial activity against S. aureus, but did not have antibacterial activity against E. coli bacteria. Endophytic fungi that had antibacterial activity were Curvularia sp., Penicillium sp., Aspergillus sp.

BackgroundTaxol 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.ResultsHigh 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.ConclusionsIn 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.

Paclitaxel (taxol), a diterpene natural compound was first extracted from the bark of yew trees. However, the method cannot meet its increasing demand on the market due to unprecedented yew cutting, low amounts of taxol production, the laborious and slow process of taxol extraction. Recently, efforts have been made to develop alternative means of taxol production. Microbial fermentation would be a promising method in the production of taxol at industrial scale. Fungal endophytes have the capacity to produce bioactive compounds and can independently synthesize secondary metabolites similar to those made by the host plants. Optimization of the fermentation culture is one of the most important strategies in increasing taxol production by endophytic fungi supplemented with several substances including carbon sources, nitrogen sources, precursors, inducer and the metabolic bypass inhibitors. Improved fermentation techniques and different biotechnological strategies such as gene cloning, gene transformation, mutations are widely being used on endophytic fungi in order to increase the productivity of the taxol. In this review, the different strategies used to produce taxol from endophytic fungal biotechnology have been discussed.

Pogostemon cablin is the medicinal plants that produces patchouli essential oils as secondary metabolites which has multiple functions including antibacterial ability. The secondary metabolites in plants mostly associated with their endophytic fungi. In this study we isolated endophytic fungi from Pogostemon cablin’s leaves and examined antibacterial activity of the endophytic fungi against Escherichia coli and Staphylococcus aureus, as well as find out the identity of most potential isolate based on Internal Transcribe Spacer (ITS) region. The isolation of endophytic fungi was performed using surface sterilization method on Malt Extract Agar (MEA) medium. The antibacterial activity was tested using paper disc on Muller Hinton Agar (MHA) medium and molecular identification was amplified using ITS 4 and ITS 5 primers. The isolation process resulted in 5 isolates of endophytic fungi. The antibacterial assay indicated one potential isolate with the highest antibacterial activity when tested against E. coli and S. Aureus, exhibited 20.9 mm and 19 mm clear zone respectively. Molecular identification from ITS region database depicted that the potential isolate has high homology with Nigrospora sp. by 99% similarity. This result suggested that the antibacterial ability of essential oils from the Pogostemon cablin’s leaves might has high correlation with the occurrence of endophytic fungi.

Trizelia, Winarto. 2016. Diversity o endophytic entomopathogenic fungus from cacao (Theobroma cacao). Pros Sem Nas Masy Biodiv Indon 2: 277-281. The endophytic fungus is a fungus that is associated with healthy host tissues without causing disease symptoms. This fungus can be developed as the biological control agents of cacao pests. This study aimed to isolate, selected and evaluate the potency of endophytic fungi from cacao crops (Theobroma cacao) as entomopathogen. Endophytic fungi were isolated from leaves, branches and fruit of cacao. Screening endophytic fungal isolates pathogenic to insect were conducted using fifth instar larvae of Tenebrio molitor. The results showed that the number of isolates of endophytic fungus isolated from the cocoa plant more found on the leaves (45.61%) compared to the isolated from the branch (19.30%) and cacao fruit (35.09%). The results of the research also showed that of the 57 isolates of endophytic fungi that were successfully isolated from cacao, there are 20 isolates (35.09%) are pathogenic in insects that caused mortality of T. molitor larvae above 20%. Endophytic fungi from cacao were pathogenic in insect and can be developed as bioinsecticides identified as Beauveria sp., Aspergillus sp., and Fusarium sp.