An endophytic Basidiomycete, Grammothele lineata, isolated from Corchorus olitorius, produces paclitaxel that shows cytotoxicity
An endophytic Basidiomycete, Grammothele lineata, isolated from Corchorus olitorius, produces paclitaxel that shows cytotoxicity
Avizit Das 1 2
Mohammad Imtiazur Rahman 1 2
Ahlan Sabah Ferdous 1 2
Al- Amin 1 2
Mohammad Mahbubur Rahman 0 2
Nilufar Nahar 2
Md. Aftab Uddin 2
Mohammad Riazul Islam 1 2
Haseena Khan 1 2
0 Development of Fiber and Polymer Science Laboratory, BCSIR, Dhaka, Bangladesh, 3 Department of Chemistry, University of Dhaka, Dhaka, Bangladesh, 4 Department of Genetic Engineering and Biotechnology, University of Dhaka , Dhaka , Bangladesh
1 Department of Biochemistry and Molecular Biology, University of Dhaka , Dhaka , Bangladesh
2 Editor: Vijai Gupta, Tallinn University of Technology , ESTONIA
Grammothele lineata, an endophyte isolated in our laboratory from jute (Corchorus olitorius acc. 2015) was found to be a substantial paclitaxel producer. Taxol and its related compounds, produced by this endophyte were extracted by growing the fungus in simple nutrient media (potato dextrose broth, PDB). Taxol was identified and characterized by different analytical techniques (TLC, HPLC, FTIR, LC-ESI-MS/MS) following its extraction by ethyl acetate. In PDB media, this fungus was found to produce 382.2 μgL-1 of taxol which is about 7.6 x103 fold higher than the first reported endophytic fungi, Taxomyces andreanae. The extracted taxol exhibited cytotoxic activity in an in vitro culture of HeLa cancer cell line. The fungal extract also exhibited antifungal and antibacterial activities against different pathogenic strains. This is the first report of a jute endophytic fungus harboring the capacity to produce taxol and also the first reported taxol producing species that belongs to the Basidiomycota phylum, so far unknown to be a taxol producer. These findings suggest that the fungal endophyte, Grammothele lineata can be an excellent source of taxol and can also serve as a potential species for chemical and genetic engineering to enhance further the production of taxol.
Data Availability Statement; All relevant data are within the paper
Funding: This work was supported by Subproject:
CP- 3250, window 2, academic innovation fund,
Under the Higher Education Quality Enhancement
Project (HEQEP), Funded by Ministry of Education,
Government of People's Republic of Bangladesh
with the assistance of The World Bank (funding
received by HK). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
It is generally assumed that fungal endophytes have the capacity to produce bioactive
compounds and can independently synthesize secondary metabolites similar to those made by the
host plants [
]. Endophytic fungi gained enormous attention when detection of taxol in the
endophytic fungus (Taxomyces andreanae) isolated from yew plant (Taxus brevifolia) was first
]. Paclitaxel (Taxol1), the first taxane isolated from natural sources (plant and
fungi) has proven to be effective against a broad range of cancers, generally considered to be
recalcitrant to conventional chemotherapy and is ranked the world's first billion dollar
anticancer drug [
]. However, production of taxol is still a challenge in medical and
pharmaceutical sectors. 20 kg of the bark of tropical and temperate yews (Taxus species), a leading
source of taxol is required for every mg of taxol [
]. It may be mentioned that 2.5±3.0 g of taxol
is required for a full regimen in cancer treatment [
], which makes it quite expensive and
unreachable for most people. At the same time, collection of compounds like taxol from a
plant source is a slow process and would lead to destruction of their ecological habitat
rendering conservation of such plants a necessity [
]. As such, there is a growing interest in
alternative sources of taxol. Several methods have been developed for taxol production, namely total
chemical synthesis [
], semi-synthesis from its precursor  and plant tissue cell culture
based production [
]. But the large number of reaction steps required for chemical synthesis
is difficult to pursue and extraction of precursors for semi-synthesis of taxol is costly. At the
same time, long incubation period, less biomass, low yield and genetic instability of plant tissue
cell culture based methods make all the three procedures inefficient. Since the description of
the first taxol producing fungus [
], microorganisms are being explored as potential
replacements for an environmentally acceptable, comparatively simple and inexpensive method of
taxol production [
]. Although the amount of taxol produced by endophytic fungi is relatively
small in comparison to that of plants, fast growth at high cell density cultivation and the
possibility of scale-up on an industrial level make endophytic fungi a promising alternative [
Microorganisms which are said to produce taxol are primarily but not exclusively- endophytes
of plants known to harbor some form of medicinal value [
]. Some of the taxol producing
fungi are soil borne [
] or even plant pathogens [
]. Moreover, few bacteria have also been
mentioned to yield taxol [
]. Many publications and their resulting patents have been
reported regarding the biosynthesis of taxol and related taxanes by microorganisms [
which a recent one is a taxol producing fungus from dermatitic scurf of the Giant Panda [
Jute (Corchorus sp.), an annual dicotyledonous crop is known mostly for its high quality
tensile natural fiber [
]. Although not reported to produce taxol, C. olitorius has long been
recognized as a medicinal herb and its extract is known to have apoptotic activity on tumor
cell lines [
]. In addition, C. olitorius has also been described to possess promising
antibacterial and antifungal activity [
]. A recent study has found a diverse community of endophytic
fungi in C. olitorius [
]. Further unrevealing of jute endophytes and gaining an understanding
of the antitumor activity of jute extracts framed the background of our current work. The
objective of this study was to identify by molecular, analytical, spectral and bio-assay based
methods, jute endophytic fungi having an independent capacity to produce taxol. Three jute
endophytic fungal isolates were initially found to be positive for the genes of taxol biosynthesis
pathway. One among the three is SDL-CO-2015-1, a Basidiomycete capable of producing taxol
ascertained by TLC, HPLC, LC-ESI-MS/MS and FTIR. The isolated taxol was effective against
HeLa cancer cell line and the fungal extract was found to be promising in antimicrobial
screening as well. This endofungus SDL-CO-2015-1, identified as Grammothele lineata is the first
ever Basidiomycete found to possess a capacity for taxol production.
Materials and methods
Collection of plant samples and isolation of endophytic fungi
Fresh plant samples (root, stem, leaf, flower and seed) of jute (C. olitorius) collected from the
botanical garden of the University of Dhaka, were surface sterilized by washing under running
tap water, rinsing with 70% ethanol for one min, then treating with 4% sodium hypochlorite
for three min. Finally samples were soaked in autoclaved milli-Q water and dried on sterile
filter paper [
]. The sterilized samples were then cut into small pieces using a sterile blade and
incubated on a potato dextrose agar (PDA) (HIMEDIA1) plate (Petri plate, Scientific Systems,
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India) at 28ÊC. After 7±10 days of incubation, a mixed fungal culture appeared on the plate,
from which pure and single culture plates for 25 individual fungi (data not shown) were
generated through a repeated sub-culture. All processes were carried out under sterile conditions.
Fungal DNA isolation
DNA from different fungal species was isolated using the modified SDS method [
]. In brief,
fungal mycelia were crushed with liquid nitrogen followed by addition of lysis buffer
containing 3% SDS, 50 mM Tris-Cl (pH 8.0), 50 mM EDTA and 2% mercaptoethanol. The thawed
suspension was incubated for 60 min at 65ÊC and then centrifuged for 5 min at 5000 x g
(Eppendorf Centrifuge 5810 R) to precipitate the cell debris. Supernatant was then mixed with
an equal volume of phenol: chloroform: isoamylalcohol (25:24:1) mixture and centrifuged
again for 10 min at 18,500 x g. Collection of supernatant was followed by addition of equal
volume of chloroform: isoamylalcohol (24:1) mixture and centrifuged for 10 min at 18,500 x g.
The final supernatant was then collected, 25 μL of 3 M Na-acetate was added, the volume
made 1 mL with isopropanol and kept overnight at -30ÊC. The suspension was centrifuged the
next day for 10 min at 18,500 x g to collect the DNA pellet. This pellet was dissolved in 300 μL
TE buffer and kept at 65ÊC for 15 min. Next 15 μL of 2 M Na-acetate and 100% ethanol was
added to make the volume 1mL. Centrifugation was again carried out at 18,500 x g for 10 min.
Supernatant was discarded and the pellet was washed with 70% ethanol. The pellet was finally
dried and dissolved in TE buffer. Concentration and purity of the DNAs were checked using a
PCR based molecular screening for taxol producing endophytic fungi
Primary search for taxol producing fungi was PCR based, using specific primers for three key
genes of the taxol biosynthetic pathway in a GeneAmpR PCR System 9700 (Applied
Biosystem). The genes screened were- ts encoding a rate limiting enzyme taxadiene synthase (ts-F:
ATCAGTCCGTCTGCATACGACA, ts-R: TAAGCCTGGCTTCCC GTGTTGT), dbat encoding a
10-deacetylbaccatin III-10-O-acetyl transferase (dbat-F: ATGGCTGAC ACTGACCTCTCAGT,
dbat-R: GGCCTGCTCCTAGTCCATCACAT) and bapt encoding a C-13 phenyl propanoid side
chain-CoA acyltransferase or bapt (bapt-F: CCTCTCTCCGCCATTGACAA CAT, bapt-R:
]. For a reaction volume of 15 μL 50 ng of DNA sample
was used together with, 0.33 μM of specific primers, 1X Taq buffer, 200 μM dNTPs and
0.375U Taq DNA polymerase. Positive samples i.e. samples with distinct amplicons for the
different primer sets after electrophoresis in 1% agarose gel were then subjected to gel extraction
using the PureLink™ Quick Gel Extraction Kit (Invitrogen, Germany) followed by single pass
sequencing (1st Base Laboratories, Malaysia). Sequence data were analyzed using BLAST
Identification and characterization of fungal isolate
One of the fungal isolates SDL-CO-2015-1 positive in genetic screening, was then subjected to
macroscopic, microscopic and molecular identifications.
Morphological characterization of the fungus. For macroscopic observation, the fungus
was grown on 90 mm disposable petri plates containing PDA. The fungal culture was
monitored continuously from the day of inoculation until the plates were fully covered with mature
mycelia and spores.
Microscopic study was carried out with fungal parts stained with lactophenol aniline blue
(Sigma, Germany), 5% KOH solution and observed under an inverted fluorescent microscope
(EVOS FL, ThermoFisher Scientific, USA).
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Molecular identification of SDL-CO-2015-1. For molecular identification, ITS (internal
transcribed spacer region) primers, ITS-1 (50-TCCGTAGGTGAACCTGCG-30) and ITS-4:
(50TCCTCCGCTTATTGATATGC-30) which amplify the highly variable ITS1 and ITS2 sequences
surrounding the 5.8S-coding sequence and situated between the Small Sub-Unit-coding
sequence (SSU) and the Large Sub-Unit-coding sequence (LSU) of the ribosomal operon were
used . PCR amplicons were analyzed in the same way as mentioned earlier. ITS sequence
of the endophytic fungi was used to estimate phylogenetic relationship by the maximum
likelihood method using the MEGA software (version 7.0, Biodesign Institute, USA). Bootstrap
analyses were based on 1000 replicates to assess the level of confidence of each node in the
gene tree [
Preparation of fungal extracts and isolation and identification of taxol
Quantification of fungal taxol
Different concentrations of standard paclitaxel solutions were analyzed in HPLC in the same
way as mentioned above for the construction of a standard curve. The peak areas of different
concentrations of the standard solutions were used to quantify fungal taxol per liter of total
HPLC purified fungal and standard taxol were first dissolved in 10% DMSO in Dulbecco's
modified Eagle's medium (DMEM). HeLa cells were cultured in a 40 mL culture flask with
DMEM supplemented with 1% penicillin-streptomycin (1:1), 0.2% gentamycin and 10% fetal
bovine serum (FBS) and incubated at 37ÊC with 5% CO2 . After 24 hr incubation cells
were recovered from the culture flask by discarding the media followed by washing with PBS
and incubated with 2 mL of 0.25% 1X trypsin-EDTA for 5 min at 37ÊC with 5% CO2. Next the
cells were collected into a 50 mL falcon tube and centrifuged at 500 x g for 5 min. The resulting
pellet was dissolved in 2 mL PBS and the cell number was counted in a hemocytometer using a
trinocular microscope with a camera (Olympus, Japan). Cells (1x 104 cells per well) in DMEM
were seeded onto the wells of a 96-well plate and incubated 24 hr at 37ÊC with 5% CO2. Then
100 μL of purified taxol and standard taxol each at a concentration of 0.005 μM and 10%
DMSO in DMEM (used as a vehicle control) were applied and incubated at 37ÊC with 5%
CO2. After 24 hr the cells were collected, washed with PBS, stained with PI (propidium iodide)
and analyzed with a fluorescence activated cell cytometer (FACs). Duplicates were used for
both the sample and the standard.
Antimicrobial activities of both extra- and intracellular extracts (prepared according to the
method mentioned above) were assayed against indicator bacterial and fungal strains by well
diffusion assay. In this study, two pathogenic fungal strains, Macrophomina phaseolina and
Aspergillus fumigatus and two bacterial strains, gram positive Staphylococcus aureus and gram
negative Burkholderia sp were used as indicator strains. 100 μL of the indicator bacterial and
fungal suspensions were spread on tryptic soya agar (TSA) and PDA plates respectively. 6 mm
diameter wells were then made in each plate with the help of a borer. 40 μL of different
concentrations (Tables 1 and 2) of either intra or extracellular extracts of SDL-CO-2015-1 were loaded
onto each well. The plates were then incubated for 24 hr at 37ÊC for antibacterial assay and 36
hr at 28ÊC for antifungal assay. The tests were done in duplicates.
Two independent replicates were taken into account for each test. Results are given as
means ± standard deviation. Statistical analyses were done using one way ANOVA and
Tukey's test in R program (alpha value 0.05) with P value <0.001 considered as highly
significant. ` ' denotes P<0.001. For each individual indicator organism, means that do not share a
letter are significantly different.
A total of 25 fungal isolates were screened initially for the presence of taxol biosynthetic
pathway genes, ts, dbat and bapt. DNA sequences of the genes available in the public database for
fungi and plant were used in designing and synthsizing gene specific primers in order to find
potential candidates. After PCR amplification, fungal isolate, SDL-CO-2015-1 was (with two
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*** denotes p value <0.001.
For each individual indicator organism, means that do not share a letter are signi®cantly different.
Negative Control (no extract)
Zone of inhibition in mm
others, data not shown) found to be PCR positive for ts and dbat gene (Fig 1A) (sequences
provide Table A in S1 File). In our quest for a taxol producing endophyte it was decided to
advance further with SDL-CO-2015-1.
Morphological characterization of PCR positive fungal isolate, SDL-CO2015-1
Surface color and texture of SDL-CO-2015-1 colonies were found to be white and cottony on a
PDA plate (Fig 2A and 2D). These colonies were yellowish white on the reverse side (Fig 2B),
widely effused, strongly adnate with the media surface, latitudinal spreading 650±750 μm thick
(Fig 2C) with reticulate furrows; occurring in a teeth pattern, or in labyrinth form (Fig 2E),
growing over the edge of the petri dish and becoming dark with age. No change in color was
*** Denotes p value <0.001.
For each individual indicator organism, means that do not share a letter are signi®cantly different.
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Fig 1. Screening for genes of taxol biosynthetic pathway and molecular identification of the fungal isolate. (A)
SDL-CO2015-1 shows a specific band of about 650bp approximately the expected size for both ts and dbat genes amplicons obtained specific
primer pairs were used (M = 1kb+ Ladder, 1 = PCR amplicon for ts gene, 2 = PCR amplicon for dbat gene) in 1% agarose gel. (B)
PCR amplicon for the ITS specific region used for molecular identification of the fungal isolate SDL-CO-2015-1 in 1% agarose gel
(M = 1kb+ Ladder, 3 = PCR amplicon for ITS region) (for the sequence see Table A in S1 File).
observed when dry. The intermediate layer of context was composed of densely gelatinized
interwoven hyphae, opaque in nature. No zonate or radiate pattern was observed. Skeletal
hyphae were found to be predominant, containing holobasidia (Fig 2G), basidiospores were
2.8 μm (Fig 2G upper-left corner) in size and cylindrical in shape. Septa were observed
between the cells (Fig 2F, red arrow), filaments were found to be long, less branched with
2.5~3 μm thickness (Fig 2H). Basidia clavate was found to be tetraspored.
Molecular identification and phylogenetic analysis of SDL-CO-2015-1
After isolation of fungal DNA, PCR with ITS (Fig 3A) specific primers gave a sharp and single
band of approximately 650 bp (Fig 1B). The amplicon was then sequenced (Table A in S1 File).
An online BLAST search of the sequence exhibited similarity with several species of the genus
Grammothele and some uncultured endophytic fungi, where 99% identity was observed with
Grammothele lineata (query coverage 97%). Phylogenetic relationship was determined
through alignment and cladistic analysis of the homologous nucleotide sequences among the
fungal species (Fig 3B). According to the evolutionary distance and morphological characters,
SDL-CO-2015-1 was identified as Grammothele lineata belonging to the Polyporaceae family
from the Basidiomycota phylum.
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Fig 2. Macroscopic and microscopic characterization of the fungal isolate. 20 day old PDA plate showing growth of endofungus
SDL-CO-2015-1, (A,B) surface and reverse of the colonies respectively (C) lateral section of texture, (D) cottony texture on PDA plate, (E)
reticulate furrows in teeth pattern, (F) septum (red arrow) and dikaryotic hyphae (blue arrow), (G) holobasidia spore (upper-left corner), (H)
low branching hyphae.
Isolation and identification of taxol in extracts of Grammothele lineata SDL-CO-2015-1
HPLC was used to isolate and purify taxol from a 21 day old culture of Grammothele lineata
SDL-CO-2015-1. Presence of taxol in the fungal extract was first identified by TLC comparing
Fig 3. Phylogenetic tree. (A) 5.8S-coding sequence situated between the Small Sub-Unit-coding sequence
(SSU) and the Large Sub-Unit-coding sequence (LSU) of the ribosomal operon. (B) Based on sequence
homologies of the ITS region, SDL-CO-2015-1 was found to have identity with Grammothele lineata.
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with standard taxol. Extracellular extract of SDL-CO-2015-1 gave a band similar to standard
taxol (Rf value both fungal and standard taxol was found to be 0.56) and displayed a dark grey
color when sprayed with 1% vanillin/sulfuric acid (w/v) (Fig 4).
A characteristic peak with almost similar retention time as standard taxol was obtained for
the extracellular extract of the fungus in HPLC, in a multistep gradient solvent system. The
fungal taxol was next purified through semi preparative HPLC. It again exhibited the same
retention time which was 31.3 min as the standard taxol (Figure A in S1 File).
LC-ESI-MS/MS scan of extracellular extract of SDL-CO-2015-1 gave a characteristic
molecular ion peak (M+H)+ similar to taxol at m/z 854 (Figure B in S1 File). To confirm the presence
of taxol, multiple reaction monitoring (MRM) was also performed using a triple quadrupole
LC-MS/MS system, which monitors both the taxol ions and the characteristic fragment peaks
of the precursor. We obtained daughter ion peaks at m/z 286, 367, 395, 464, 509, 545, 551, 568
and 587. These peaks were identical to standard taxol in multi reaction monitoring (MRM)
(Fig 5). Both LC-ESI-MS/MS and LC-MRM-MS data appear to attest the presence of taxol in
the fungal extract [
The FTIR spectral data of fungal taxol from SDL-CO-2015-1 gave peaks at 3484.8 and
3393.2 cm-1 for hydroxyl (±OH) and amide (±C (O) NH±) group stretches respectively (Fig 6).
Aliphatic CH stretch was observed at 2928.8 cm-1 and ester and ketone group (C = O) stretches
were observed in the region of 1729.1 and 1741 cm-1 respectively. The aromatic ring (C = C)
stretching frequency was observed in the region of 1667 cm-1. TheÐCOO- stretching
frequency was observed at 1371.29. A peak observed at 1073 cm-1 was due to the presence of
aromatic C, H bond [
In order to quantitate the amount of taxol produced by the endofungus, extraction from a
21 day old fungal culture in PDB media, was carried out using the protocol described in the
methods section. The characteristic peak of fungal taxol at 31.367 min in HPLC was used to
quantitate the amount of taxol produced by the fungus. Data obtained for the area of the peaks
vs standard taxol concentrations were used to construct a standard curve (Figure C in S1 File)
for estimating the amount of the amount of taxol produced by our endofungus. The yield of
taxol from one liter of PDB medium was calculated to be 382.2 μgL-1
Cytotoxic activity of purified fungal taxol
HPLC purified fungal taxol was tested for cytotoxic activity by apoptotic assay on HeLa cell
line using PI staining. In FACs analysis 35% cell death was observed for both standard and
fungal taxol (at a concentration of 0.005μM) whereas 12.64% cell death was observed for 10%
DMSO in DMEM media (Fig 7).
Antimicrobial activity of fungal extract
Antimicrobial activity was tested for an extract of Grammothele lineata SDL-CO-2015-1
against some pathogenic fungi and bacteria. Antifungal activity was assessed against two fungi,
one plant pathogen, Macrophomina phaseolina and one opportunistic infectious pathogen,
Aspergillus fumigatus. Only intracellular extract of SDL-CO-2015-1 gave a clear zone of
inhibition against both the fungi (Figure D in S1 File) (Table 1). Antibacterial activity was assessed
against a gram-positive (S. aureus) and a gram negative bacteria (Burkholderia sp). Clear zones
of inhibition were found for both intracellular and extracellular extract against the both
indicator strains (Figure D in S1 File) (Table 2). For each indicator strain the assays for two
independent events were replicated twice.
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Fig 4. Thin layer chromatographic analysis of SDL-CO-2015-1 extract along with the standard taxol.
TLC analysis of fungal taxol along with standard taxol in 1% vanillin/sulfuric acid (w/v). Standard taxol and
fungal taxol show similar Rf value (0.56).
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Fig 5. Analysis of taxol fragmentation pattern in LC-MRM-MS. (A) LC chromatogram for standard taxol. (B) MRM of standard taxol
show characteristic peaks at m/z 286, 367, 395, 464, 509, 545, 551, 568 and 587. (C) LC chromatogram of fungal extracellular extract. (D)
MRM of fungal taxol showed similar characteristic m/z with standard taxol at 286, 367, 395, 464, 509, 545, 551, 568 and 587.
Fig 6. FTIR spectral analysis of fungal taxol.
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Fig 7. Induction of apoptosis in HeLa cell line treated with standard taxol, fungal taxol and 10% DMSO in DMEM media; as
determined by PI staining through FACs analysis. (A) Untreated cells without PI staining (vehicle only). (B) Untreated cells with PI
staining (vehicle only). (C) Cells treated with 0. 005μM of standard taxol. (D) Cells treated with 0. 005μM of HPLC purified fungal taxol. (E)
Bar diagram shown as a FACs profile, standard taxol was used as a positive control and 10% DMSO in DMEM media as a vehicle control.
The standard and fungal taxol gave almost same percentage of cell death which is significantly more than the vehicle control. These assays
were performed for two independent events and replicated twice. (*** denotes p value<0.001, indicating high level of significance).
Plant endophytic fungi are recognized as an important and novel resource of natural bioactive
products. They offer a number of compounds with notable anti-tumorigenic applications [
that are part of the war against cancer. Taxol, diterpene in nature, is among the most popular
natural compounds that has been effectively used in cancer treatment as well as in
neurodegenerative and polycystic kidney disorders [
]. So far, researchers have found more than 20
genera of endophytic fungi that produce taxol isolated from both Taxus and non-Taxus species
and the wide range proves that both taxol-producing fungi and their hosts have considerable
biological diversity [
]. With the help of genetic screening and chemical analyses we isolated
an endophytic fungus, SDL-CO-2015-1 identified as Gramothele lineata harboring a taxol
producing capacity from a jute plant, Corchorus olitorius. The isolated fungal taxol was also tested
for the efficacy of its anticancer activity against HeLa cancer cell line.
A major significance of this study is the first ever identification of a fungus from the
Basidiomycota phylum that is able to produce taxol. Till 2013, about 46 genera and 111 species of
endophytic fungi producing antitumor components have been reported and taxonomically,
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nearly all of them belong to Ascomycota (96%) and only a paltry 3% have been found from the
Basidiomycota phylum [
]. Interestingly almost all fungi reported to produce taxol are under
the phylum Ascomycota, however, G. lineata, isolated as a jute endophyte belongs to
Basidiomycota phylum and is the first ever taxol producing fungi to be reported from this phylum.
Basidiomycetes are known to produce terpenoids as their primary class of secondary
metabolites and often have unique structures not observed elsewhere in the natural world. On the
other hand Ascomycota are mostly known for their polyketides and non-ribosomal peptides
]. Several terpenoids from the Basidiomycota phylum show selective antitumor activity
making them excellent candidates for cancer therapy [
]. Even though this phylum is
considered the most sophisticated form of fungi, their diterpene biosynthetic pathways are largely
unknown compared to Ascomycota and are predicted to be more diverse, complex and
expected to yield more secondary compounds [
]. Basidiomycetes present a unique
opportunity for the discovery of novel terpenoid biosynthetic routes leading to the development of
compounds with new bioactivities . Thus, the ability of SDL-CO-2015-1 to produce taxol
does not come as a surprise and it is possible that the largely untapped fungal kingdom may
reveal more members capable of producing diverse bioactive compounds-including
antimicrobial compounds as found in this study. No previous work has reported the taxol producing
ability of Basidiomycetes although a review has made such a claim [
] without providing any
specific data to support the same.
Only one species under the genus Grammothele,ÐG. fuligo has been identified as an
endophyte of oil palm [
]. Our isolate, SDL-CO-2015-1 was identified to be G. lineata based on
ITS rDNA gene sequence and phylogenetic analysis. Phylogenetic analysis puts the isolate in
the same clade with other G. lineata strains. Interestingly, this clade also includes some
uncultured fungi which is not unexpected since a considerable number of fungi belonging to the
Basidiomycota phylum frequently show poor growth under laboratory conditions [
According to the features described by Reck and Rosa [
], the basidiospores of Grammothele
lineata are 2.5±3 μm thick matching with the basidiospores of our strain, SDL-CO-2015-1.
However, our strain has a rare cylindrical shape rather than the more common ellipsoidal
form. Macroscopically the surface texture and color of SDL-CO-2015-1 were found to be the
same as that described for G. lineata by others [
]. Even though not much information is
available on the morphology of G. lineata and the existing data are somewhat ambiguous,
classical morphological identification together with molecular analysis suggests SDL-CO-2015-1
to be a new strain of G. lineata.
Biochemical techniques and spectrometric analyses used for the detection of taxol are
tedious and time-intensive procedures. Therefore as a first choice it is convenient to screen for
the presence of paclitaxel biosynthetic genes. A couple of reports have used such molecular
approaches for the purpose of screening fungi [
]. However, the biosynthetic pathway
and the regulatory mechanism of taxol in fungi are still unknown. An intriguing question is
whether the pathway is conserved among fungi and plants since at this stage, genes or
pathways related to fungal taxol biosynthesis are still at large . In this study genetic screening
was carried out using three different genes that are vital to taxol biosynthesis (ts, dbat and
bapt). Although ts is a rate limiting enzyme in the till known pathway both dbat and bapt are
more diagnostic because more than ten enzymatic steps are required for the synthesis of taxol
after ts [
]. Sequence information of these genes from fungi is scant, the reliance therefore is
on taxol biosynthetic gene sequences of plant origin . As a consequence primers used for
screening taxol genes in endophytes are primarily based on available plant sequences. This
appears to be justified if we take into consideration the recent genome analysis of the
taxolproducing endophytic fungus Penicillium aurantiogriseum [
] which reveals sequence
homology of key genes involved in taxol biosynthesis between plant and fungi. Origin of this pathway
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in these two physically associated groups appear to have been facilitated by horizontal gene
transfer, HGT. However, another group has claimed to find no similarity in the pathways for
taxol biosynthesis in fungi and plants when whole genome sequence and transcriptom analysis
of two endophytic fungi from T. brevifolia were made. They envisage the biosynthetic pathway
of taxol in endophytic fungi may have a distinctly different evolutionary pattern compared to
]. Xiong, et al., 2013 have reported the ability of a specific fungus to produce taxol
although the sequence they deposited for genes of the corresponding pathway gave negligible
query coverage. Only the primer sequences were found to match [
]. However, the same
fungus was found to produce taxol as confirmed by HPLC and LC-MS. This may explain why
DNA sequence of individual genes of SDL-CO-2015-1 amplified with primers designed from
conserved regions of corresponding taxol biosynthetic genes of plants yielded inconclusive
results in in silico hybridization (blastn).
In this study, the presence of fungal taxol was screened primarily by TLC. Extracts of
SDL-CO-2015-1 was found to possess an Rf value of 0.56, congruent with standard taxol and
gave a spot dark grey in appearance when sprayed with 1% vanillin/ sulphuric acid. HPLC
analysis of the extract gave a peak in reverse phase C18 column, with almost the same retention
time as the standard taxol. Fragmentation pattern of fungal taxol in LC-ESI-MS/MS using
LC-MRM-MS was found to be identical to the standard taxol [
]. Characteristic peak at m/z
545 and 587 are possibly for 10-deacetylbaccatin-III and baccatin-III respectively which are
the main precursors of taxol, [
] attesting again the taxol production capability of the jute
endofungus. In a simple nutrient media this fungus was found to produce approximately
382.2 μgL-1 of taxol, much higher than that reported for the endophytic fungus, Taxomyces
andreanae in PDB [
]. This isolate capable of producing a considerable amount of taxol in a
simple PDB media is expected to produce more if grown in M1D media supplemented with
When the isolated taxol was tested for its cytotoxicity, it was found to be relatively
significant against the HeLa cancer cell line. G. lineata SDL-CO-2015-1 extract was also found to
have both antibacterial and antifungal activity. It is not clear at this point in time if such
bioactivities can be attributed to taxol. However, the antimicrobial activity of G. lineata is the first
ever to be observed, and leads to the expectation that this Basidiomycete will actually become a
new source of naturally effective bioactive compounds.
S1 File. Sequences of G. lineata, analytical data for fungal taxol identification, and
bioassays of fungal extracts against indicator organisms.
The authors would like to thank the Bangladesh Council for Scientific and Industrial Research
(BCSIR) DFPL (Dhaka Fiber and Polymer Science) laboratory for their services. The authors
also concede Mucosal Immunology and Vaccinology Laboratory, Infectious Disease Division
International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) for providing
facilities for FACs analysis and Intertek Bangladesh Ltd for allowing use of their facilities.
Conceptualization: HK AD MRI.
Data curation: AD.
14 / 17
Formal analysis: AD MRI.
Funding acquisition: HK.
Methodology: AD MIR AA MMR.
Project administration: HK.
Resources: HK NN MAU.
Validation: AD MRI.
Visualization: AD ASF MRI.
Writing ± original draft: AD.
Writing ± review & editing: AD ASF MRI HK.
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