• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1372-1380
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• 2013

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 $66{\mu}g/l$ 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.109-114
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• 2005

Ion fluxes across the plasma membrane activated by 1 mM $Ce^{4+}$, cell apoptosis and taxol biosynthesis in suspension cultures of Taxus cusp/data were studied. The extracellular pH sharply decreased upon the addition of 1 mM $Ce^{4+}$, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular $Ca^{2+}$ concentration decreased within the first 3 h after the addition of $Ce^{4+}$, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a $Ca^{2+}$-channel blocker indicated that the dynamic changes in extracellular pH and the $Ca^{2+}$ concentration resulted from the $Ce^{4+}$-induced activation of W uptake and $Ca^{2+}$ influx across the plasma membrane via ion channels. A pretreatment of the ion channel blocker initiated $Ce^{4+}$-treated cells to undergo necrosis, and the prior addition of the $Ca^{2+}$-channel blocker inhibited $Ce^{4+}$-induced taxol biosynthesis and apoptosis. It is thus inferred that W uptake is necessary for cells to survive a $Ce^{4+}$-caused acidic environment and is one of the mechanisms of $Ce^{4+}$-induced apoptosis. Furthermore, the $Ca^{2+}$ influx across the plasma membrane mediated both the $Ce^{4+}$-induced apoptosis and taxol biosynthesis.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.427-440
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• 1998

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.29-33
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• 2000

To develop an efficient propagation method for yew tree, zygotic embryos were cultured under various conditions. When dissected embryos were cultured on GA$_3$ containing media, the highest germination frequency was observed on WPM medium contaning 1.0 mg/L GA$_3$. For germination of the embryos, two different conditions were compared; culturing embryos with endosperm (Method I), and 2) culturing embryos only (Method II). Maximum germination was achieved in 0.5 mg/L GA$_3$ when embryos with endosperm were cultured on the media. Of the media tested, White and WPM medium were the most suitable on germination of embryos. The abnormality of yew embryos found was observed when it cultured on GA$_3$ or culture media. About 40% of the precociously germinated embryos could be developed into full seedlings. Seedlings contained taxol in high quantity (535 $\mu\textrm{g}$/g dry weight). In vitro techniques will be sewed as a useful tool for the development of transformed root cultures and biosynthesis studies.

• BMB Reports
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• v.40 no.6
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• pp.861-869
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• 2007

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of $\beta$-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.

• Journal of Microbiology and Biotechnology
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• v.29 no.10
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• pp.1570-1579
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• 2019

The fungal products dibenzodioxocinones promise a novel class of inhibitors against cholesterol ester transfer protein (CEPT). Knowledge as to their biosynthesis is scarce. In this report, we characterized four more dibenzodioxocinones, which along with a previously described member pestalotiollide B, delimit the dominant spectrum of secondary metabolites in P. microspora. Through mRNA-seq profiling in $g{\alpha}1{\Delta}$, a process that halts the production of the dibenzodioxocinones, a gene cluster harboring 21 genes including a polyketide synthase, designated as pks8, was defined. Disruption of genes in the cluster led to loss of the compounds, concluding the anticipated role in the biosynthesis of the chemicals. The biosynthetic route to dibenzodioxocinones was temporarily speculated. This study reveals the genetic basis underlying the biosynthesis of dibenzodioxocinone in fungi, and may facilitate the practice for yield improvement in the drug development arena.

• Korean Journal of Pharmacognosy
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• v.27 no.3
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• pp.262-266
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• 1996

Undifferentiated callus were induced from the young leaves of Taxus cuspidata by treatment with various combinations of plant growth hormones. The effects of light and culturing temperature on production of baccatin III, the precursor of taxol, were studied. The contents of baccatin III in the cultured callus were analysed by HPLC. The illumination of fluorescence and administration of isoleucine, one of the possible substrates in biosynthesis of terpenoids, to the culturing media increased the production of baccetin III.

• BMB Reports
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• v.40 no.5
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• pp.625-635
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• 2007

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semi-quantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.