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W
(paclitaxel) using endophyte
biotechnology
Souvik Kusari
1
, Satpal Singh
2
, and Chelliah Jayabaskaran
2
1
Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry
and Analytical Chemistry, TU Dortmund, Otto-Hahn-Str. 6, D-44221 Dortmund, Germany
2
Department of Biochemistry, Indian Institute of Science (IISc), Bangalore 560012, Karnataka, India
Taxol
W
(generic name paclitaxel) represents one of the
most clinically valuable natural products known to man-
kind in the recent past. More than two decades have
elapsed since the notable discovery of the rst Taxol
W
-
producing endophytic fungus, which was followed by a
plethora of reports on other endophytes possessing simi-
lar biosynthetic potential. However, industrial-scale Tax-
ol
W
production using fungal endophytes, although
seemingly promising, has not seen the light of the day.
In this opinion article, we embark on the current state of
knowledge on Taxol
W
biosynthesis focusing on the chem-
ical ecology of its producers, and ask whether it is actually
possible to produce Taxol
W
using endophyte biotechnol-
ogy. The key problems that have prevented the exploita-
tion of potent endophytic fungi by industrial bioprocesses
for sustained production of Taxol
W
are discussed.
Taxol
W
, an interesting case in point for revisiting
endophyte biotechnology
We recently highlighted the current bottlenecks in exploit-
ing a promising group of microbes called endophytic micro-
organisms (or endophytes) using red biotechnology, which
are capable of producing pharmaceutically-relevant second-
ary metabolites [1]. On the one hand, we are increasingly
gaining a deeper understanding of how endophytes engage
in bi-, tri-, and multipartite interactions with their host
plants as well as with other associated organisms (fungi,
bacteria, or viruses) andendosymbionts, under the selection
pressures of various biotic (such as pathogens and feeders)
and abiotic factors (such as precursors of plant/endophyte
secondary metabolites and environmental conditions) in
order to produce certain value-added natural products.
On the other hand, we have failed to translate these
amazing discoveries into successful industrial bioprocesses
for sustained production of desirable compounds using en-
dophyte biotechnology. We present the example of the
blockbuster drug Taxol
1
(generic name paclitaxel) to dis-
cuss the trade-offs of attempting to translate potent endo-
phytic Taxol
1
producers into industrial microbial factories.
Chemical ecology of taxane production by yew plants
Taxol
1
, belonging to a class of complex diterpenoids called
taxanes and possessing an unusual oxytane ring together
with a tricyclic core, is a blockbuster anticancer drug.
Being unique in its mode of action of halting the prolifera-
tion of cancer cells [2,3], the drug was approved by the US
FDA to treat a variety of tumors including breast, ovarian,
and AIDS-related Kaposis sarcoma, among others. It was
originally isolated from the bark of Pacic Yew, Taxus
brevifolia [4]. By and large, the production of Taxol
1
and other taxanes is conned to a narrow taxonomic group
of higher plants belonging to the genus Taxus (family
Taxaceae, syn. Coniferales). Among the other four genera
of this family, namely Amenotaxus, Autrotaxus, Pseudo-
taxus, and Torreya [5], only Autrotaxus [6] and Pseudo-
taxus [7] contain some simpler taxanes. Among the conifers
apart from the genus Taxus only two other species have
been reportedly claimed to produce taxanes. These include
a close cousin of Taxus called Cephalotaxus (Cephalotax-
aceae) [7] and also Podocarpus gracilor Pilger (Podocarpa-
ceae) [8]. With the exception of the genus Taxus, the
occurrence of this diterpenoid in other reported gymno-
sperm taxa has not been extensively studied and seems
conned to only an exceptionally limited number of species.
Interestingly, the presence of paclitaxel and other taxanes
has also been shown in an angiosperm from the family
Betulaceae, namely Corylus avellena L. [9,10]. Although
the molecular basis of taxane production in this angio-
sperm has not received adequate attention (see [11] for a
recent report on the transcriptome analysis of a Taxol
1
-
producing endophyte harboring this plant), it might imply
either an independent evolution of Taxol
1
biosynthesis in
unrelated taxonomic groups or some type of evolutionary
relatedness in the chemical ecology of these different taxa.
From the nigh-exclusive occurrence of this class of
molecules in yew and the evolutionary and taxonomic
uniqueness of Taxaceae and more importantly Taxus,
Opinion
0167-7799/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.03.011
DOI of companion article: http://dx.doi.org/10.1016/j.tibtech.2014.03.009.
TRENDS in Biotechnology
Figure 1. Prevalent consensus biosynthetic route for Taxol
1
in Taxus species. Abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol-4-phosphate; GGPPS,
geranylgeranyldiphosphate synthase; TS, taxa-4(5),11(12)-diene synthase that catalyzes the committed step of this pathway; T5aH, taxa-4(5),11(12)-diene-5a-hydroxylase;
TAT, taxa-4(5),11(12)-diene-5a-ol-O-acetyltranseferase; T10bH, taxane-10b-hydroxylase; z, oxytane ring formation and branch migration enzymes including taxane 2a-O-
benzoyltransferase (T2BT or DBBT = debenzoyltaxane-2
0
-a-O-benzoyltransferase) as well as C-13 hydroxylation and steps taking pathway flux towards non-Taxol
1
-type
molecules; DBAT, 10-deacetylbaccatin III-O-acetyltransferase; BAPT, baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase; DBTNBT, 3
0
-N-debenzoyl-2
0
-deoxytaxol-N-
benzoyltransferase which follows hydroxylation in the side chain by an unknown enzyme; PAM, phenylalanineaminomutase; *, b-phenylalanine coenzyme A ligase.
Multiple arrows imply more than one biosynthetic step. The Taxol
1
biosynthetic pathway is proposed to have about 20 different enzymatic steps in Taxus plants.
Opinion
Trends in Biotechnology June 2014, Vol. 32, No. 6
308
Box 1. Sustained Taxol
W
supply using endophytes
Genetic engineering approaches can be used to engineer the Taxol
1
biosynthetic pathway (Figure IA). This includes overexpression of the
important or all of the Taxol
1
biosynthetic pathway genes and their
promoter modulation, including other complimentary genetic engi-
neering approaches such as epigenetic engineering involving tran-
scription factors or chromatin modifier elements, and gene silencing.
Taxol
1
-producing endophytes and other heterologous hosts including
simpler organisms such as yeast and bacteria could serve as ideal
candidates for such an attempt. Taking advantage of inter-organismal
effects could also lead to greater Taxol
1
/taxane yields (Figure IB). This
strategy may involve co-cultivation of two or more organisms for
elicitation of Taxol
1
biosynthesis, Taxol
1
pathway-intermediate bio-
transformation, or utilization of metabolic dead ends/high yield Taxol
1
pathway-intermediates fromone organismby another organismas the
exogenously supplied substrates, and even combinatorial Taxol
1
/
taxane biosynthesis by different organisms. Various molecular-,
biotechnological-, and bioprocess-related methodologies, tools, tech-
niques, and optimization strategies, both stand-alone or in combina-
tion, are known or proposed to affect the Taxol
1
/taxane yields of a
given endophyte or a recombinant heterologous host (Figure IC). These
would seem to prove especially relevant when used in combination
with the genetic engineering and inter-organismal combinatorial
approaches. For example, a heterologous host engineered for the
Taxol
1
biosynthetic pathway gene(s) could be optimized for an
inducible Taxol
1
production, a greater cellular release of Taxol
1
,
two-phase growth and production cycles, and silencing or modulation
of its own Taxol
1
pathway negative feedback, flux-diversionary, or
dead-end metabolite metabolic steps.
(A)
TS GGPPS T5H TAT T10H DBAT PAM etc.
Metabolic engineering of Taxol
biosynthec pathway
Heterologous hosts
Flux balance (pathway and protein engineering)
Enzyme engineering
Co-culture and culture opmizaon
Bioreactor design
Removal of metabolic bolenecks/dead ends
Strain improvement (gene overexpression,
mutagenesis, genome shuing. gene silencing,
engineering, and physiochemical opmizaon
for increased product excreon)
Superior Taxol
yields ???
Endophyc fungi
(B)
(C)
d i
TRENDS in Biotechnology
Figure I. Possible strategies for achieving sustained Taxol
1
supply using endophytes. (A) Genetic engineering approaches involving the Taxol
1
biosynthetic pathway.
(B) Inter-organismal contribution towards greater Taxol
1
/taxane yields. (C) Bioprocessing-based strategies for increasing Taxol
1
yield.
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
309
contributions from heterologous hosts and other optimiza-
tion methodologies and tools such as intracellular com-
partment optimization, storage and efux modulation, and
control of pathway regulatory elements (Box 1). However,
delineation of the molecular mechanisms of Taxol
1
bio-
synthesis and regulation thereof remains a prerequisite for
all such endeavors. Most notably, as seen from the Taxol
1
biosynthetic pathway of Taxus sp., there seems to be an
obvious hurdle in engineering such a lengthy and complex
pathway in its entirety in heterologous hosts. Transforma-
tion of genes of the entire pathway is a challenge and more
importantly, regulation of Taxol
1
production encompass-
ing epigenetic modulation and signaling crosstalk itself
remains a poorly understood topic. Taxol
1
-producing en-
dophytic fungi, therefore, still present a viable and long-
term target, despite many unanswered questions (Box 2).
Acknowledgments
Research in the laboratory of S.K. (INFU, TUDortmund) is supported in
part by the International Bureau (IB) of the German Federal Ministry of
Education and Research (BMBF/DLR), Germany, the Ministry of
Innovation, Science, Research, and Technology of the State of North
Rhine-Westphalia, Germany, the German Academic Exchange Service
(DAAD; Welcome to Africa initiative), and the German Research
Foundation (Deutsche Forschungsgemeinschaft, DFG). S.K. is a Visiting
Researcher at the Department of Plant Sciences, University of Oxford,
South Parks Road, Oxford OX1 15 3RB, United Kingdom. S.K. gratefully
acknowledges M. Spiteller for approving and authorizing, Gail M.
Preston for hosting, and TU Dortmund for supporting his stay at the
University of Oxford. Research in the C.J. laboratory (IISc Bangalore) is
supported by grants fromthe Department of Biotechnology (DBT), India
and the Council of Scientic & Industrial Research (CSIR), India. S.S.
thanks CSIR and DBT for fellowships. We thank Bhagat Singh for
assistance in preparation of Figure 1. We apologize to the numerous
investigators whose publications could not be cited here owing to space
constraints.
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Is the Taxol
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Taxus plants and what is its origin?
Why do endophytic fungi tend to lose their Taxol
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production
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Do these endophytic fungi produce Taxol
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how does that differ fromtheir in vitro production, especially with
regard to its biosynthetic regulation?
What is the molecular basis of the reported elicitation of
endophyte Taxol
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biosynthesis by host plant components?
Do the host plant Taxol
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pathway intermediates play a part in
fungal Taxol
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How much is the storage, release, and compartmentalization of
Taxol
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biosynthesis in fungi affected by metabolic flux dynamics
connecting primary and secondary metabolism or vice versa?
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