JBA-06956; No of Pages 15

Biotechnology Advances xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances

journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Endophytes as in vitro production platforms of high value plant

secondary metabolites
Aarthi Venugopalan, Smita Srivastava ⁎
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India

a r t i c l e i n f o a b s t r a c t

Article history: Many reports have been published on bioprospecting of endophytic fungi capable of producing high value bioac-
Received 9 February 2015 tive molecules like, paclitaxel, vincristine, vinblastine, camptothecin and podophyllotoxin. However, commercial
Received in revised form 27 June 2015 exploitation of endophytes for high value-low volume plant secondary metabolites remains elusive due to widely
Accepted 16 July 2015
reported genomic instability of endophytes in the axenic culture. While most of the endophyte research focuses
Available online xxxx
on screening endophytes for novel or existing high value biomolecules, very few reports seek to explore the
Keywords:
possible mechanisms of production of host–plant associated or novel secondary metabolites in these organisms.
Endophytic fungi With an overview of host–endophyte relationship and its possible impact on the secondary metabolite
Host–endophyte relationship production potential of endophytes, the review highlights the evidence reported for and against the presence
Secondary metabolite of host-independent biosynthetic machinery in endophytes. The review aims to address the question, why
In vitro production should and how can endophytes be exploited for large scale in vitro production of high value phytochemicals?
Bioprocess optimization In this regard, various bioprocess optimization strategies that have been applied to sustain and enhance the prod-
Axenic culture instability uct yield from the endophytes have also been described in detail. Further, techniques like mixed fermentation/
Mixed fermentation
co-cultivation and use of epigenetic modifiers have also been discussed as potential strategies to activate cryptic
Epigenetic modifiers
gene clusters in endophytes, thereby aiding in novel metabolite discovery and overcoming the limitations
associated with axenic culture of endophytes.
© 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. What are endophytes? — definition, discovery and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Host–endophyte relationship — role in host plant evolution, defense and secondary metabolite production . . . . . . . . . . . . . . . . . . . . 0
3.1. Production of plant secondary metabolites in endophytes — the possibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Using endophytes as in vitro production platforms for plant secondary metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4.1. Limitations of producing bioactive secondary metabolites from plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4.2. Alternative methods for in vitro production of plant secondary metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4.3. Host-independent biosynthesis of plant secondary metabolites in endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4.3.1. Case studies on taxol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4.3.2. Case studies on camptothecin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5. Strategies for sustainable and enhanced production of secondary metabolites in endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.1. Strain improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.1.1. Mutagenesis in endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.1.2. Methods for genetic transformations in endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.2. Bioprocess optimization strategies for enhanced secondary metabolite production in endophytes . . . . . . . . . . . . . . . . . . . . . 0
5.2.1. Optimization of fermentation parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.2.2. Elicitor/inhibitor addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.2.3. Precursor feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5.2.4. Use of adsorbent resins/solid supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

⁎ Corresponding author.
E-mail address: smita@iitm.ac.in (S. Srivastava).

http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
0734-9750/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
2 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx

6. Strategies to unlock the cryptic/silenced gene clusters in endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6.1. Co-cultivation and mixed fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
6.2. Use of epigenetic modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction
Fungi have been known to be a rich repository of medicinally
important compounds since the discovery of penicillin. Today, the
range of drugs derived from fungi stretch from antibiotics to immune-
suppressants to anti-cholesterol drugs (statins). While plants still
remain the major source of drugs or their lead molecules, with every
new bioactive molecule reported from a plant source, there follows re-
ports of endangered status or even extinction of a medicinally impor-
tant plant due to over-harvesting. Hence, the focus turned toward
fungi namely the “endophytes”, which reside within these medicinally
important plants and thus may have acquired their medicinal abilities.
The landmark in this area of endophyte bioprospecting was undoubted-
ly the discovery of Taxomyces andreanae (Stierle et al., 1993), the first

Fig. 1. Structures of various bioactive molecules produced by endophytes. (a) Taxol, (b) camptothecin (c), vincristine (d), vinblastine (e), huperzine (f), podophyllotoxin (g), diosgenin and
(h) azadirachtin.

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx 3

endophyte reported to produce taxol, the billion dollar anti-cancer lead
molecule (Fig. 1a). The number of patents and publications that follow-
ed this discovery triggered high hopes of a sustainable alternative
production route using endophytic fungi for almost all the major
life-saving drugs, without having to harvest the trees to meet the
ever-increasing market demands. Indeed the initial discovery was
followed by a plethora of different endophytes reported to produce
various other bioactive molecules including camptothecin, vincristine,
vinblastine, huperzine, podophyllotoxin, diosgenin and azadirachtin
(Zhao et al., 2011b) (Fig. 1b–h). However, despite numerous reports
documenting the secondary metabolites from endophytes in the last
two decades (Table 1), there have been no major breakthroughs in
terms of commercial exploitation of any endophyte as a source of bioac-
tive molecules. Reports related to inconsistent production of many of
these biomolecules in the axenic culture of endophytes upon isolation
from the host plant have raised doubts on commercial feasibility
of endophytes as sustainable production platforms. In this review,
literature evidence available in support as well as in opposition of the
presence of host-independent biosynthetic machinery in endophytic
fungi has been presented. Recent advances in the bioprocessing
of endophytes post the taxol boom have also been discussed. This re-
view aims at highlighting the need for greater research on the complex
ecological interactions between endophytes and their host plant as well
as with other endophytic microbiota in the host. A deeper understand-
ing of these interactions, when combined with rational bioprocess
optimization, will enable the provision of a culture environment
which is conducive to sustainable and maximum production of high
value secondary metabolites, during in vitro cultivation of potential
endophytes.
2. What are endophytes? — definition, discovery and classification
The term endophyte was coined by Heinrich Anton de Bary in 1884 to
originally define any organism occurring within plant tissues (Hyde and
Soytong, 2008). Broadly this definition includes bacteria (Kobayashi and
Palumbo, 2000), fungi (Stone et al., 2000), algae (Trémouillaux-Guiller
et al., 2002), insects (Tooker and Hanks, 2004) and other vascular plants
(Marler et al., 1999). However, majority of the endophyte research has fo-
cused on endophytic fungi (Arnold and Lewis, 2005), almost making
them synonymous with the term ‘endophyte’. These endophytes have
been differentiated from mycorrhizae based on the three hallmarks of a
mycorrhizal association which includes the absence of a localized inter-
face of specialised hyphae (present in most mycorrhiza), the absence of
synchronized plant–fungus development, and the lack of plant benefits
with respect to nutrient transfer (Brundrett, 2004).
The earliest records of the presence of endophytic fungi have come
from the 400 million year old fossils of the early Devonian Rhynie
chert deposits (Krings et al., 2007) which suggest that endophyte–
plant associations may have evolved along with the evolution of higher
plants. Modern day studies on endophytic fungi can be traced back to
mid-19th century. Based on differences in evolutionary relatedness,
taxonomy, plant hosts, and ecological functions, the endophytic
fungi are classified as the clavicipitaceous or grass endophytes
(C-endophytes) and the non-clavicipitaceous endophytes (NC-
endophytes) of non-vascular plants, ferns, conifers, and angiosperms
(Rodriguez et al., 2009), with the latter being further separated into
three functional classes based on host colonization patterns, mechanism
of transmission between host generations, in planta biodiversity levels,
and ecological function. Interest in the NC-endophytes has now

Table 1
Examples of high value secondary metabolites isolated from endophytes.

Compound Bioactivity Endophyte strain Referencea

Azadirachtin A and B Natural pesticide Eupenicillium parvum Kusari et al. (2012b)

Berberine Antibiotic, anti-diabetic, anti-inflammatory, anti-cancer. S6 Gao et al. (2008)
Camptothecin Anti-cancer Several strains Zhao et al. (2010)
Cytochalasins Anti-cancer, antibiotic Rhinocladiella sp., Strobel and Daisy (2003)
Digoxin glycoside Cardiotonic, diuretic, anti-epileptic Several strains Kaul et al. (2012)
Diosgenin Progesterone precursor, cholesterol lowering activity Cephalosporium sp. Zhao et al. (2010)
Paecilomyces sp.
Emodin Anti-diabetic, anti-viral, anti-cancer Thielavia subthermophila Zhao et al. (2010)
INFU/Hp/KF/34B
Gentiopicrin Anti-malarial fungicide and larvicide QJ16 and QJ18 Yin et al. (2009)
Ginkgolide B Used in treatment of cardiovascular diseases, cerebrovascular diseases Fusarium oxysporum SYP0056 Cui et al. (2012)
and migraines
Gymnemagenin Anti-diabetic Penicillium oxalicum Parthasarathy and Sathiyabama (2014)
Huperzine A Potential treatment for neurodegenerative diseases Several strains Zhao et al. (2010)
Hypericin Anti-depressant, anti-inflammatory, anti-microbial, anti-oxidant, anti-viral T. subthermophila Zhao et al. (2010)
INFU/Hp/KF/34B
Kaempferol Anti-oxidant, anti-cancer Fusarium chlamydosporum Chaturvedi et al. (2014)
Lapachol Anticancer Aspergillus niger Nirupama et al. (2011)
Alternaria alternata
Piperine Antimicrobial, antidepressant, anti-inflammatory, anti-oxidative, Periconia sp. Verma et al. (2011)
anti-cancer
Podophyllotoxin Anti-cancer Several strains Zhao et al. (2010)
Quercetin glycoside Anti-hypertensive, anti-cancer, anti-inflammatory, anti-oxidant Penicillium sp. Gh01 Nair and Padmavathy (2014)
Rhein Antitumor, anti-inflammatory, antimicrobial and hemostatic Fusarium solani R13 You et al. (2013)
Rohitukine Anti-inflammatory, immuno-modulatory, anti-cancer Fusarium proliferatum (MTCC 9690) Kumara et al. (2012)
Sanguinarine Anti-cancer F. proliferatum BLH51 Wang et al. (2014b)
Sipeimine Antitussive and expectorant Fritillaria ussuriensis Fu7 Yin and Chen (2008)
Subglutinols A and B Immune-suppressants Fusarium subglutinans Lee et al. (1995)
Tanshinone I and IIA Cardiotonic, anti-inflammatory Emericella foeniculicola TR21 Ma et al. (2011)
Trichoderma atroviride D16 Ming et al. (2012)
Taxol Anti-cancer Several strains Zhao et al. (2010); Strobel and Daisy (2003);
Strobel et al. (2001); Strobel et al. (1999)
Toosendanin Digestive tract-parasiticide, selective presynaptic blocker, effective O-L-5, O-SC II-4, O-RC-3 Zhao et al. (2010)
anti-botulismic agent; agricultural insecticide
Vincamine Vasodilator, cerebral stimulant XM-J2 Yin and Sun (2011)
Vincristine & vinblastine Anti-cancer Several strains Zhao et al. (2010)
a
A single representative reference for each compound is provided — more examples exist.

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
4 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
increased due to their diversity, ecological roles, potential applications
and the ability to switch between endophytic and other lifestyles.
3. Host–endophyte relationship — role in host plant evolution,
defense and secondary metabolite production
All the plant species, including non-vascular plants, ferns, conifers
and angiosperms, are believed to be in symbiotic relationship with
endophytic fungi (Rodriguez et al., 2009). Many plants harbor as
many as hundreds of endophytes within apparently healthy tissues
(Stone et al., 2000). The co-evolution of fungi and land plants may
have begun much earlier than the early Devonian period. It has been
hypothesized that the early mutualistic symbiosis between an alga
and fungal partners catalyzed the evolution of the modern land plants
from their aquatic ancestors with the fungal partner enabling the
survival of the ancestors of land plants against drought, mineral
deficiencies, UV radiation and temperature fluctuations which charac-
terized the early Paleozoic land (Selosse and Le Tacon, 1998). Indeed,
endophytic fungi are known to confer upon their host plant adaptation
to stresses like salinity, tolerance to drought, metals, disease, heat, and
herbivory, and also promote growth via biosynthesis of plant hormones
and nutrient acquisition (Rodriguez et al., 2009). For example, the
symbiotic dependence of Dichanthelium lanuginosum plants on their
endophytic Curvularia sp. has been reported for thermoprotection, and
that of the grass species Leymus mollis with its endophyte Fusarium
culmorum has been reported for salinity tolerance (Rodriguez et al.,
2009), with the endophyte also benefitting due to the symbiotic
association in both cases.
One of the tools used by the endophytic fungi for aiding in the
survival of their host plants is the production of secondary metabolites.
Two theories have been proposed to explain how endophytes may be
assisting the host plants in chemical defense (Kusari et al., 2012a). The
mosaic theory (Carroll, 1991) proposes that endophytes create a
heterogenous chemical composition within and among otherwise ge-
netically uniform plant organs thus varying their worth for herbivores
or susceptibility to infection by pathogens, while a parallel theory
proposed by Arnold et al. (2003) suggests that endophytes might act
as “acquired immune systems” in their host plants. Examples of second-
ary metabolites produced by endophytes, with known role in deter-
rence of herbivory, include peramine, ergot and indole diterpene
alkaloids, lolines and swainsonine — a trihydroxyindolizidine alkaloid
(Panaccione et al., 2014). A recent report has found a direct correlation
between the variation in taxol content among different parts of a Taxus
plant and the quantity of a resident taxol producing fungal endophyte,
wherein depletion of the endophyte via treatment with fungicides re-
sulted in decreased expression of taxol biosynthesis genes in the plant
and hence in reduced plant taxol accumulation (Soliman et al., 2013).
3.1. Production of plant secondary metabolites in endophytes — the
possibility
Although endophytic fungi have been researched for more than a
century now, it is only in the last twenty years that they have been
reported as being capable of mimicking the secondary metabolite reper-
toire of the host plant. The “xenohormesis” hypothesis (Howitz and
Sinclair, 2008) states that “heterotrophs”, due to evolutionary selection
pressures, have evolved the ability to sense signaling and stress-induced
molecules from plants, with this sensing ability being retained even if
the capacity to biosynthesize these compounds is lost over time. This
lends credence to the idea that the stress signaling in plants is also
sensed by the endophytic fungi which in turn mount defense responses
similar to the host plant. Hence, it naturally follows that conserved gene
clusters would exist across plants and their endophytes, which might be
activated by suitable associations leading to the production of similar
secondary metabolites in both plant and fungi (Kusari et al., 2012a). In
order to explain how interactions with the host plant shape the
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
metabolic machinery of endophytes, the example of camptothecin
producing endophytic fungi will be discussed here.
The evolutionary role of camptothecin and its derivatives include
protecting plants against heat shock (Zu et al., 2003) and defending
them against insect and pathogen attack by binding and inhibiting
the DNA topoisomerase I (Sirikantaramas et al., 2009). While the
camptothecin biosynthetic pathway in plants has not been completely
elucidated, many of the rate-limiting enzymes have been identified
(Sun et al., 2011). In turn, a host-dependent cross species biosynthetic
pathway for camptothecin production in endophytic fungi has been
proposed by Kusari et al. (2011b), where some of the fungal biosynthet-
ic enzymes were shown to be homologous to their host counterparts
which could be the result of horizontal gene transfer (HGT) during co-
evolution. Both the host plant and its endophytes have also developed
several intrinsic as well as co-evolutionary resistance mechanisms in-
volving mutations in the Top1 gene in order to protect themselves
against endogenously produced camptothecin (Kusari et al., 2011a).
Thus, it can be said that every aspect of growth, development and
survival of the endophytes and that of their plant hosts is dependent
on their co-evolution which is characterized by a balanced antagonism,
the hallmark of endophytism (Schulz et al., 1999).
The fact that endophytic fungi are found to mimic the host plant
secondary metabolite profile also led to a possibility that these plant
metabolites could in fact be a product of their respective endophytes
(Kusari et al., 2012a). This has been demonstrated in the case of
ansamitocin, a potent antitumor maytansinoid originally isolated from
plants belonging to Celastraceae, Rhamnaceae, and Euphorbiaceae
families and later from the actinomycetes Actinosynnema pretiosum
ssp. pretiosum and A. pretiosum ssp. auranticum. Although initially
isolated from the shrub Maytenus serrata, unlike other plant metabo-
lites, detailed characterization of the ansamitocin biosynthetic gene
clusters has been carried out only in the actinomycete A. pretiosum
ssp. auranticum rather than in the plant by Yu et al. (2002). Further,
the lack of evidence for the production of maytansinoids from cultured
plant cells (callus and suspension) and the absence of a key
maytansinoid biosynthetic gene in plants substantiated the possibility
that the true biosynthetic source of the maytansinoid backbone could
be a bacterial endophyte and symbiosis could have triggered the
production in plants (Cassady et al., 2004).
4. Using endophytes as in vitro production platforms for plant
secondary metabolites
4.1. Limitations of producing bioactive secondary metabolites from plants
Several statistics point at how indispensable plants are as secondary
metabolite sources. Twenty five percent of the drugs prescribed world-
wide originate from plants, with plant-based drugs accounting for 11 %
of the 252 basic and essential drugs recognized by the WHO (Dubey
et al., 2012). At least 120 distinct plant-based active compounds are
being used as important drugs in one or more countries (Taylor,
2000). Natural products, especially plant-derived, have contributed to
47% of the anticancer drugs in the market (Newman and Cragg, 2007).
However, the indispensability of plant-based bioactive molecules unfor-
tunately brings with it several disadvantages — (i) extremely low yields
which vary with environmental conditions, (ii) limited supply leading
to over-harvesting, and (iii) extremely complex structures which
make total synthesis and semi-synthesis very challenging. The current
demand–supply statistics for various high value phytochemicals are
listed in Table 2. Over-harvesting of trees to meet the increasing market
demand has led to their endangerment and even extinction in some
cases. While the tropical rainforests, which are some of the richest and
yet untapped sources of high value metabolites, are rapidly plummeting
on one hand from 14% to a mere 6%, less than 1% of these trees and
plants have been subject to bioprospecting so far, essentially implying
that hundreds of valuable medicines could be lost to us forever before
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx 5

being discovered (Taylor, 2005). Hence, search for alternative and
sustainable sources of these high value plant secondary metabolites is
the need of the hour.
4.2. Alternative methods for in vitro production of plant secondary
metabolites
In vitro plant cell/tissue cultures, including callus and hairy root
cultures, have been extensively reviewed for mass production of plant
secondary metabolites (Mulabagal and Tsay, 2004; Srivastava and
Srivastava, 2007). However, limitations with plant cell/tissue culture
technology include genomic instability, low yields, and scale-up difficul-
ties resulting in only few success stories at the industrial level, like taxol
production by Phyton biotech and production of shikonin, ginseng and
berberine by Mitsui Chemicals (Linden, 2006).
Total chemical synthesis, another alternative, has been explored
for most of the important metabolites with success in some cases
(e.g. vanillin — most of its demand is met through chemical synthesis)
and not so in most other cases due to the sterical complexity (e.g. mor-
phine has five chiral centers, which makes its chemical synthesis
complex and uneconomical) and structural complexity (e.g. chemical
synthesis of paclitaxel involves 40 reactions with harsh solvent and
low product yield which makes its total synthesis impractical)
(Wilson and Roberts, 2012).
The discovery of endophytic fungi capable of mimicking the second-
ary metabolite repertoire of their host in most of the medicinally or oth-
erwise industrially valued plants has given an impetus to the possibility
of using microbial fermentation to meet the ever-growing demand for
several life-saving drugs. Moreover, endophytes have been known to
evolve novel resistance mechanisms to the toxic metabolites produced
by them and the host as a co-evolutionary adaptation (Kusari et al.,
2011a; Mu et al., 1999), which simplifies bioprocessing of these organ-
isms without the risk of growth inhibition due to self-toxicity of the
product.
Microbial fermentation has certain inherent advantages that make
this strategy more robust than others. The culture medium for fungal
cells is relatively simple and inexpensive, largely consisting of industrial
by-products/wastes like corn steep liquor and molasses, whereas plant
cell culture medium requires expensive additives like phytohormones
which increase the production cost. Faster growth rate of fungi makes
the fermentation period shorter with lesser risk of contamination in
comparison to that with slow growing plant cells/tissue cultivations.
Optimization, regulation of fermentation parameters and subsequent
scale-up is relatively easier in microbial fermentations in comparison
to that in plant cell/tissue cultivations in bioreactors. Moreover, micro-
bial cultures are easily amenable to yield enhancement strategies like
strain improvement, precursor feeding, elicitor addition, appending in-
hibitors and co-cultivation (Zhao et al., 2010). Microbial fermentation of
endophytes can thus relieve the threat of over-harvesting of plants
while ensuring a steady supply of important plant secondary metabo-
lites, irrespective of environmental conditions.
4.3. Host-independent biosynthesis of plant secondary metabolites in
endophytes
While there are many reports available on the isolation of endophyt-
ic fungi capable of producing high value plant derived drugs, no break-
throughs have been reported yet on the application of these strains for
the commercial production of high value phytochemicals. A widely ac-
knowledged (and reported) problem of subsequent attenuation in the
product yield with subculture of endophytes under axenic conditions
is one of the major limitations in the commercialization of this
bioprocess. Scientific investigators have questioned the existence of
host-independent biosynthetic machinery in endophytes due to the in-
consistent production of secondary metabolites observed in vitro. On
the contrary, sustainable production of metabolites like tanshinone IIA
and taxol by the axenic culture of endophytic fungi up to 10 generations
has also been reported (Ma et al., 2011; Zhao et al., 2013b). Thus, there
exists evidence for both supporting the claims of a host-independent
machinery and for opposing it. Three schools of thought exist on the or-
igins of secondary metabolism in plants, all of which emphasize on the
role of endophytes — 1.) Both plants and endophytic microbes may have
co-evolved parallel pathways to produce these natural products;
2.) Horizontal gene transfer (HGT) may have taken place between
plants and microbes during their long period of association; 3.) Either
plants or endophytic fungi may produce these secondary metabolites
and transfer them to the other symbiont (Karuppusamy, 2009). Further,
the xenohormesis hypothesis supports the idea that endophytes might
have the machinery to synthesize the host plant metabolites (Kusari
et al., 2012a). Recent studies have reported the presence of key

Table 2
Demand–supply statistic for various high value metabolites derived from plants.

Compound Uses Demand and supply statistics Reference

Taxol Antitumor • Annual sales in 2006 = 3.7 billion USD Malik et al. (2011)
• Annual demand = 600 kg
• Treatment per patient consumes about eight 60-year-old yew trees
• 10,000 kg of Taxus bark or 3000 yew trees = 1 kg of taxol
Camptothecin Antitumor • Supplied from two plants — Camptotheca acuminata and Nothapodytes foetida — both critically endangered Patwardhan (2006)
• ~1000–1500 tons of wood chips = 1 ton of camptothecin
• Annual sales in 2003 = 1 billion USD
• Annual biomass demand in India in 2006 = 500–700 metric tons
• Annual biomass demand in Japan in 2006 = 1000 metric tons
• Global camptothecin demand in 2011 required 100 million trees
Vinca alkaloids Antitumor • Very low yield from plant (0.0003% DW) Hendrawati et al.
• Annual demand = 0.3 tons (2012)
• Annual sales = 200 million USD
• Production via plant cell cultures unsuccessful so far
Digitalis Cardiotonic • Annual demand = 1000 metric tons plant material Mangathayaru (2013)
glycosides • Patients require 1 mg/day
• Worldwide drug demand = several thousand kilograms per year
• India — annual demand ≥30 quintals
Diosgenin Progesterone • Accounts for 2/3rds of the total world consumption of steroids Dangi et al. (2014)
precursor • Annual demand = 3000 tons
• India — annual demand = 150 tons.
• India total production = 30 tons
Podophyllotoxin Antitumor • Only 2 plant sources known — Podophyllum hexandrum and Podophyllum peltatum — both endangered Alam et al. (2009)
• Annual demand ≥100 metric tons
• Annual supply = 50–80 tons

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
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6 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
biosynthetic genes in taxol and camptothecin producing endophytic
fungal strains (Kusari et al., 2011b, 2014). Whole genome sequencing
of an endophytic Shiraia sp. Slf14, previously reported to produce both
huperzine A and hypocrellin A, has also revealed the presence of a puta-
tive huperzine A (HupA) biosynthetic gene cluster (Yang et al., 2014a).
These findings provide strong indications that endophytic fungi are
secondary metabolite factories by themselves. To further elaborate on
the various contradictory evidences available, the taxol and
camptothecin producing endophytic fungi have been discussed below
as useful case-studies.
4.3.1. Case studies on taxol production
The list of taxol producing endophytic fungi is an ever-growing one,
with variable taxol yields (from nanogram to milligram per liter media)
and loss in productivity with subculture. This has prompted some
workers to question whether the source of the fungal taxol is in fact
the host plant pool. However, the isolation of these endophytes from
non-taxol producing trees has also been reported (Flores-Bustamante
et al., 2010; Li et al., 1998a), which contradicts the idea of fungal taxol
being a host plant adduct. In addition, de-novo synthesis of paclitaxel
in axenic culture of a fungal endophyte was demonstrated by Stierle
et al. (1993) by feeding of [1–14C] acetic acid and L-[U-14C] phenylala-
nine as precursors. Several studies have reported the presence of taxol
biosynthetic genes like ts, dbat and bapt in these endophytes using
primers designed based on published sequences from Taxus trees. A
comprehensive list of taxol genes identified in endophytes has been
documented by Kusari et al. (2014). In most cases, the fungal genes
identified have been found to be homologous to their plant counter-
parts (with N96% sequence similarity) (Staniek et al., 2009; Zhang
et al., 2009a, b; Zhou et al., 2007), which strongly supports the horizon-
tal gene transfer (HGT) theory.
However, the sheer complexity of the taxol biosynthetic pathway,
which involves more than 20 steps localized to different subcellular
compartments with genes possibly scattered over different plant
chromosomes, has raised doubts over the possibility of transfer of the
entire pathway from the host plant to the endophyte (Heinig et al.,
2013). Further, the occurrence of horizontal gene transfer between
distantly-related organisms is a rarity, constrained by the amount of
genetic information transferred and other genetic barriers, which
contradicts the observation of taxol biosynthesis in many different
endophytic fungi (Kurland et al., 2003). However, some studies offer
support to the idea that the microbial taxol gene cluster exists indepen-
dent of the plant genes having co-evolved with the plant machinery
rather than being acquired through HGT. This has been well established
in the case of gibberellin biosynthetic pathways in fungi and higher
plants which show several differences and have evolved independently
rather than by HGT (Hedden et al., 2001; Tudzynski, 2005). Similar find-
ings have been reported in case of taxol producing endophytes where
the fungal ts and bapt genes have been found to have low similarities
to the corresponding plant genes (Xiong et al., 2013; Yang et al.,
2014c), which suggests and supports co-evolution theory rather than
HGT theory.
In a detailed study conducted on various taxol producing endophyte
strains including T. andreanae, Heinig et al. (2013) found no fungal
genomic sequence related to known taxane-specific sequences from
yew trees, thus ruling out the possibility of horizontal gene transfer. In
order to test the other possibility, which is the existence of an indepen-
dently evolved biosynthetic pathway, shotgun sequencing of the full
genome of the strains T. andreanae and EF0021 was done, revealing
only certain putative terpene synthases which were neither similar to
each other nor homologous with plant terpene synthases. However, a
significant degree of sequence similarity would be expected of enzymes
that catalyze reactions as complex as taxadiene synthesis even if the
evolution of the plant and fungal biosynthetic pathways happened
independently. Hence, the authors concluded that fungal taxane was
in fact residual plant taxane which, due to its lipophilicity, formed
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
complexes with the endophyte cell wall structures that would persist
for up to a few passages of the fungal cultures. However, a very recent
study reported by Yang et al. (2014c) on the whole genome sequencing
of taxol producing endophyte Penicillium aurantiogriseum NRRL 62431
directly contradicts the findings of Heinig et al. (2013). When the fungal
genome was scanned via multiple sequence alignments for potential
paclitaxel biosynthesis genes, putative genes showing low homology
with the Taxus genes but with distinct conserved amino acid sites
emerged, supporting the independent evolution theory over HGT. Out
of 13 known paclitaxel genes, only 7 potential homologs were identified
in NRRL 62431, which supports the divergence of the two biosynthetic
pathways with conservation of only specific enzyme sites important
for the activity rather than the whole protein structure. The
P. aurantiogriseum strain NRRL 62431 continued to produce taxol for
more than twelve passages without any contact with the host plant,
thus ruling out the possibility of passive taxane accumulation in fungal
cell walls. Moreover, the isolation of taxol producing endophytes from
non-taxol producing plants like Citrus medica and Taxodium distichum
leaves the debate open on how these endophytic fungi acquired the
ability to produce taxol independently.
4.3.2. Case studies on camptothecin production
The key enzymes identified in the biosynthetic pathway of
camptothecin so far include tryptophan decarboxylase (TDC), geraniol
10-hydroxylase (G10H), secologanin synthase (SLS) and strictosidine
synthase (STR). In the camptothecin-producing endophytic fungal
strain of Fusarium solani INFU/Ca/KF/3, Kusari et al. (2011b) reported
the presence of TDC, G10H and SLS genes by using primers designed
from corresponding plant gene sequences. These fungal genes were
highly homologous with their plant counterparts. However, no amplifi-
cation product was obtained with any STR gene-specific primer and so it
was argued that the endophyte might be using the host STR pool for
camptothecin biosynthesis. However, neither artificial reconstitution
into the host nor supplementing with various host plant tissue extracts
could restore the camptothecin biosynthesis in the strain which showed
complete product attenuation after seven generations of subculture. To
explain the yield attenuation observed upon sub-culturing, the strain in
its seventh generation subculture was probed for the presence of the
biosynthetic genes. While STR was not detected, TDC, G10H and SLS
gene products were obtained, but the homology with the plant counter-
parts was much lesser indicating random mutations. It was therefore
proposed that the lack of in planta selection pressure under axenic
conditions caused immediate degradation of the partial camptothecin
biosynthetic machinery possessed by the fungus, thus completely
impairing its ability to synthesize camptothecin even when the host–
endophyte association was restored. Contrary to these observations,
there exist reports on other endophytic strains that have been shown
to sustain camptothecin production in axenic cultures up to several
generations (Pu et al., 2013).
When whole genome sequences of 53 fungi (including plant and
animal fungi) were analyzed, there was no detection of STR or any
other terpene indole alkaloid (TIA) pathway downstream genes includ-
ing those involved in vincristine and vinblastine synthesis or even the
taxol biosynthesis genes (Sachin et al., 2013). This finding is supported
by studies on the evolution of STR and strictosidine synthase-like pro-
teins (SSLs), where the latter has been reported from plants, algae and
cyanobacteria but rarely from fungi (Sohani et al., 2009). Three different
hypotheses have been proposed by Sachin et al. (2013) to explain
camptothecin production by endophytic fungi and attenuation on sub-
culturing with specific reference to STR. The first hypothesis is that
STR function in fungi is carried out by a new SSL or a totally different
protein, which would explain why conventional attempts at detecting
STR gene or its orthologs would fail. If such a gene did exist, both the
production of camptothecin and its attenuation could be explained,
the latter due to gene silencing or methylation process under axenic
conditions. The second hypothesis argues for the presence of the STR
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
gene on certain extra-chromosomal elements (ECEs) in the fungal cyto-
plasm while the third hypothesis proposes that the STR gene is carried
in a plasmid within endohyphal bacteria with the genes being acquired
via HGT in both cases. Product attenuation in these two cases is ex-
plained by the loss of the ECEs including plasmids and the endohyphal
bacteria upon repeated sub-culturing of the axenic fungal culture. Re-
ports on the presence of plasmids carrying secondary metabolic gene
clusters in antibiotic producing Streptomyces species give strong support
to the latter two hypotheses (Medema et al., 2010; Mochizuki et al.,
2003). However, evidences from endophytes to prove these hypotheses
are yet to emerge.
5. Strategies for sustainable and enhanced production of secondary
metabolites in endophytes
In order to realize and completely tap the secondary metabolite ar-
senal of endophytic fungi on an industrial scale, yield and productivity
enhancement strategies at several levels are required. A combination
of genetic/metabolic and bioprocess engineering may be used to sustain
and enhance production of high value secondary metabolites from
endophytic fungi. While the potential of endophytes as sources of high
value phytochemicals and novel natural products is enormous, the
yields are insufficient to be commercially exploited. Hence, the develop-
ment of high yielding endophytic fungal strains (via strain improve-
ment techniques) which are able to synthesize metabolites at
commercially feasible levels is required. These improved strains can
further be subjected to various bioprocess optimization strategies
well known in microbial fermentations for further enhancement in
secondary metabolite yield and productivity.
5.1. Strain improvement
Strain improvement involves not just product yield enhancement
but also improvement of other characteristics including removal of un-
wanted co-metabolites, improved utilization of inexpensive carbon and
nitrogen sources and alteration of cellular morphology for improved ox-
ygen transfer in the fermenter or for ease in downstream processing
(Barrios-Gonzalez et al., 2003). Two strategies are used either separate-
ly or in concert — 1) mutagenesis followed by random/rational
screening and protoplast fusion techniques and 2) genetic engineering
that includes secondary metabolite gene cluster amplification, gene
disruption, and cloning of regulatory genes.
5.1.1. Mutagenesis in endophytes
This technique involves the use of mutagens to induce changes in
the genetic characteristics of organisms. Mutagens employed may be
physical (ultraviolet (UV), X-ray, γ-rays, laser, microwave, ion beams
etc.) or chemical (ethyl methyl sulfonate (EMS), nitrosoguanidine
(NTG), sodium nitrite (NaNO2), diethyl sulfate (DES)). Selection of
improved mutants is done either through random screening for the
desired trait or through rational screening which involves selection for
a particular characteristic of the desired genotype that is different
from the one of final interest (Barrios-Gonzalez et al., 2003)
e.g. hygromycin resistant mutants are frequently selected to screen fur-
ther for increased taxol production (Kai et al., 2009). Compound muta-
tions are also induced by using two or more mutagens in order or using
only one mutagen but repeatedly or using two or more mutagens at the
same time in order to utilize the interaction between mutagens (Zhou
et al., 2010). While the exact mechanism behind the observed increase
in the production of secondary metabolites in microbes upon mutation
is unclear, genetic studies indicate that mutation may be causing chang-
es in regulatory genes, thereby leading to phenotype changes as well as
increased production of metabolites (Adrio and Demain, 2006).
Mutagenesis in fungal endophytes can be carried out using protoplasts,
mycelium or spores. This strategy has been very successfully applied to
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
7
taxol producing endophytes. Table 3 lists examples of usage of muta-
genesis for increasing yield of various metabolites from endophytes.
Mutagenesis on mycelium has the disadvantage of genetic
separation in off-springs and it is difficult to control and optimize the
conditions for mutagenesis in the case of spores, while mutagenesis
on protoplasts does not have these limitations (Kai et al., 2009). Further,
the lack of a cell wall makes protoplasts more environment-sensitive
and so protoplast mutagenesis is a very useful strategy for yield en-
hancement in endophytic fungi. Most of the studies so far have focussed
on optimization of the conditions for protoplast preparation and regen-
eration as factors including pH, temperature, time, enzyme combina-
tions, osmotic stabilizers, pre-treatment, regeneration medium and
method significantly affect the success rate (Xu et al., 2006b; Zhao
et al., 2004; Zhou et al., 2008). There are a few reports on product
yield enhancement achieved via protoplast mutagenesis and regenera-
tion (Kai et al., 2009; Zhang et al., 2011b; Zhao et al., 2005b; Zhou
et al., 2005). Protoplast fusion is another technology which has been
applied to endophytes for enhancing product yields. A high-level taxol
producing strain HDF-68 was obtained by inactivated protoplast fusion
of the two mutant strains UV40–19 and UL50–6 of Nodulisporium
sylviforme (Zhao et al., 2013b). Compared to the parent strains, taxol
production increased to 468.62 μg/L i.e. 24.51% and 19.35% compared
with UV40–19 and UL50–6, respectively.
5.1.2. Methods for genetic transformations in endophytes
Most studies on the genetic engineering of endophytic fungi are
still at a preliminary stage, focusing on the development of efficient
methods for fungal DNA transformation. However, the success of
these studies provides a strong platform for future research on the
insertion of key biosynthetic and/or regulatory genes via utilization of
improved genetic transformation methods for sustaining and enhanc-
ing secondary metabolite production from the axenic culture of the
endophytes. Currently, most of the available reports on genetic manip-
ulation of endophytes are on taxol producing endophytes. Long et al.
(1998) used a protoplast transformation protocol in the endophytic
fungus, Pestalotiopsis microspora while developing molecular genetic
techniques to elucidate the fungal taxol biosynthetic pathway.
Unexpectedly, the majority of P. microspora transformants contained
extrachromosomal DNAs with terminal telomeric repeats which can
play an important role in adaptive and developmental implication. A
procedure for PEG-mediated transformation of the taxol producing en-
dophytic fungal strain Ozonium sp. EFY-21 was established by Wei et al.
(2010). Yamin et al. (2012) successfully used this protocol to construct a
fungal expression vector (pV2+-TS-pAN7-1) containing the taxadiene
synthase gene (ts), encoding for a rate-limiting enzyme in taxol
biosynthesis pathway, and used it to generate a five times higher
taxol-yielding mutant of the endophyte, Ozonium sp. EFY-21. Liu et al.
(2013) established an Agrobacterium tumefaciens mediated genetic
transformation (ATMT) method for a taxol-producing fungus, Ozonium
sp. EFY21, which greatly enhanced the transformation efficiency com-
pared to the traditional PEG-mediated protoplast transformation. A
similar ATMT protocol was developed for the genetic transformation
of a taxol-producing Cladosporium cladosporioides strain MD2 (Zhang
et al., 2011a). Yet another method of transformation, restriction
enzyme-mediated integration (REMI) was used for transformation of
a taxol-producing endophytic Ozonium sp. strain BT2 (Wang et al.,
2007). Compared to conventional transformation methods, REMI
increases transformation rate and the frequency of single-copy integra-
tion events (Bӧlker et al., 1995; Manivasakam et al., 2001). Spiering et al.
(2005) have reported the application of gene silencing technique like
RNAi in fungal endophytes.
Apart from taxol producing endophytes, other microorganisms too
have been utilized for taxol production via gene overexpression.
Examples include preparation of taxadiene by overexpression of genes
encoding isopentenyl diphosphate isomerase, geranylgeranyl diphos-
phate synthase, and taxadiene synthase in Escherichia coli (Ajikumar
8 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx

Table 3
Examples on usage of mutagenesis for improvement in product yield from endophytes.

Product Wild-type strain Mutant strain Mutagen Yield enhancement Reference

Taxol HD1-3 UN25-3 UV, NTG and UV + NTG 1.4 fold Kai et al. (2008)
Taxol HD1-3 UD (14–1) UV + DES 1.34 fold Zhao et al. (2011e)
Taxol Nodulisporium sylviforme HQD33 N. sylviforme NCEU-1 UV, EMS, 60Co (60Co-γ-ray) and NTG 2.5 fold Zhou et al. (2001)
Taxol Fusarium maire strain Y1117 F. maire K178 UV + DES 8.6 fold Xu et al. (2006a)
Berberine S6 S-NU-302 UV, X-rays and NaNO2 170% Gao et al. (2008)
Tanshinone IIA Emericella foeniculicola TR21 E. foeniculicola NU152 UV + NaNO2 1.46 fold Ma et al. (2011)
Vincamine XM-J2 XM-UL-7-5 UV-laser 2.1 fold (62.3% increase in biomass) Yin and Sun (2011)

et al., 2010) and expression of eight taxoid biosynthetic genes in yeast
from episomal vectors (Dejong et al., 2006). However, endophytic
fungi from the host plant offer certain advantages over other
microorganisms where genetic manipulation for product enhancement
is concerned. Specifically, the sheer complexity of biosynthetic pathways
(e.g. taxol biosynthesis involves almost 20 enzymatic reactions) makes
expression of all the genes in a foreign microorganism a very tedious
process. On the other hand, the existence of at least partial biosynthetic
enzyme machinery in endophytes makes the genetic manipulation of
endophytic fungi easier.
5.2. Bioprocess optimization strategies for enhanced secondary metabolite
production in endophytes
Use of various bioprocess optimization strategies for yield enhance-
ment of secondary metabolites has been successful with plant cell/
tissue cultivations and has been extensively reviewed (Chattopadhyay
et al., 2002; Mulabagal and Tsay, 2004; Ramachandra Rao and
Ravishankar, 2002). Strategies like media manipulation, culture
condition optimization, elicitor addition, precursor feeding and use of
adsorbent resins have been adopted in axenic cultures of endophytic
fungi for enhancing the yield of high value secondary metabolites.
Some of these strategies used in literature are discussed below.
5.2.1. Optimization of fermentation parameters
The importance of providing the right combination of culture condi-
tions for enhancing the production of secondary metabolites via
fermentation technology is well known. By optimizing and controlling
fermentation parameters like medium composition, pH, temperature,
agitation, and photoperiod, a sustainable, economic and environment
friendly bioprocess with consistent product quality and quantity
independent of the environmental variations can be developed for
industrial scale-up. Bode et al. (2002) coined the term “one strain
many compounds” (OSMAC), to describe how a single microbial strain
can be induced to produce many compounds by simply varying the cul-
ture parameters. In some cases, the OSMAC approach has even led to the

Table 4
Examples of culture parameter optimization for improvement in product yield from endophytes.

Product Strain Optimized parameters Methodology Yield enhancement Reference

Taxol Fusarium mairei UH23 Initial pH, inoculation volume, working Single factor 10.2% Dai and Tao (2008)
volume, temperature, carbon and
nitrogen source, fermentation period
Taxol Mutant F. maire K178 Nitrogen source and trace elements Plackett Burman design 1.3 fold Xu et al. (2006a)
and RSM
Taxol Nodulisporium sylviforme pH, temperature, agitation rate, Single factor 1.15 fold (including Zhao et al. (2011d)
UV40–19 fermentation period elicitors)
Taxol Pestalotiopsis microspora Monobasic sodium phosphate Single factor 2.2 fold (at 1 μg/mL) Li et al. (1998b)
Ne32
Palmarumycin C13 Berkleasmium sp. Dzf12 Metal ions RSM 6 fold Mou et al. (2013)
Palmarumycin C13 Berkleasmium sp. Dzf12 Carbon and nitrogen sources Plackett Burman design 2.5 fold Zhao et al. (2013a)
and RSM
Exo polysaccharide (EPS) Berkleasmium sp. Dzf12 Carbon and nitrogen sources, trace Fractional factorial design 6.29 fold Li et al. (2012a)
elements and RSM
Zofimarin Xylaria sp. Acra L38 Carbon and nitrogen sources Orthogonal array design, 8 fold Chaichanan et al.
Plackett Burman design (2014)
and RSM
Beauvericin Fusarium redolens Dzf2 Carbon and nitrogen sources, initial pH Plackett Burman design 1.27 fold Xu et al. (2010)
and RSM
Huperzine Colletotrichum Temperature, pH, agitation rate, Single factor 28.58% Zhao et al. (2013c)
gloeosporioides ES026 fermentation period
Carbon source 51.89%
Berberine Mutant strain S-NU-3-2 Carbon and nitrogen sources, L16 (43) orthogonal 47.2% Hong and Yang
illumination condition, temperature design (2010)
Mycoepoxydiene Phomopsis sp. Hant25 Complex media — Czapek yeast Single factor 10.3 fold Thammajaruk et al.
autolysate broth (CzYB), Malt Czapek (2011)
broth (MCzB) and modified M1D
medium (MM1D); shaking/static period
Camptothecin Fusarium oxysporum NFX06 Glucose, peptone and MgSO4 RSM 1.02 fold Musavi et al. (2014)
Camptothecin Trichoderma atroviride Medium composition, fermentation time, Single factor 50 to 75 fold (including Pu et al. (2013)
LY357 pH, temperature, agitation rate elicitor and adsorbent
addition)
Camptothecin Entrophospora infrequens Medium composition Single factor 503 ± 25 μg/100 g dry cell Amna et al. (2012)
RJMEF001 mass (in Sabouraud broth)
Vincamine XM-J2 Medium composition and pH L16 (43) orthogonal 1.4 fold (1.5 fold increase Yin and Sun (2011)
design in biomass)
Sipeimine Fritillaria ussuriensis Fu7 Medium composition and temperature L16 (44) orthogonal design 77% increase (155% Yin and Chen (2011)
increase in biomass)

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx 9

Table 5
Examples of elicitation strategy for improvement in product yield from endophytes.

Product Organism Elicitor Yield enhancement Reference

Taxol Nodulisporium sylviforme UV40–19Serine, SA, silver nitrate, ammonium acetate 1.15 fold (including culture parameters) Zhao et al. (2011d)
Taxol Periconia sp. Serinol, p-hydroxy benzoic acid, β-resorcyclic acid, 8 fold (0.01 mM benzoic acid) — all Li et al. (1998a)
gallic acid, Benzoic acid other elicitors had positive effect
Camptothecin Trichoderma atroviride LY357 Salicylic acid 3.4 fold Pu et al. (2013)
Methyl jasmonate 2.2 fold
Ca2+, Cu2+, Mn2+ Slight increase
Li+ Slight decrease
10-Hydroxy camptothecin Xylaria sp. Ce3+, Cr3+, La3+, MJ, SA, Cu2+, Fe2+, Se5+, Mn2+, 2.7 fold (0.1 mM SA) — except MJ all Liu et al. (2010)
Ca2+, Li+ elicitors had positive effect
Palmarumycin C13 Berkleasmium sp. Dzf12 Yeast extract 3.2 fold Zhao et al. (2011c)
Palmarumycin C13 Berkleasmium sp. Dzf12 Water extracted polysaccharide from host plant 2.69 fold Li et al. (2012b)
(Dioscorea zingiberensis) rhizomes
Palmarumycin C12 Berkleasmium sp. Dzf12 Crude oligosaccharide prepared by acid hydrolysis 9.83 fold Li et al. (2012c)
Palmarumycin C13 of the water extracted polysaccharide 3.24 fold
Huperzine Colletotrichum gloeosporioides ES026 Ethanol and methanol 51.89% (with ethanol) Zhao et al. (2013c)

discovery of novel metabolites (Grond et al., 2002; Wang et al., 2014a),
thus highlighting the crucial role of culture parameter optimization in
microbial fermentation for desired results. Optimization of medium
and fermentation parameters can be done either by changing one factor
at a time or by varying several factors at the same time and looking for
interactions using statistical analysis. Statistical design of experiments is
an organized approach that gives more reliable information per
experiment as it enables the visualization of the interactions among
experimental variables, leading to predictions of the response data in
areas not directly covered by experimentation (Thiry and Cingolani,
2002). Both statistical as well as single factor optimization studies
have been reported in the fermentation of endophytic fungi. Examples
of culture parameter optimization carried out in endophytic fungi for
increasing the product yield have been listed in Table 4.
5.2.2. Elicitor/inhibitor addition
Elicitors are signaling molecules which trigger the formation of
secondary metabolites in cell cultures by inducing plant defense, hyper-
sensitive response and/or pathogenesis related proteins (Baldi et al.,
2009). They are classified as biotic and abiotic based on their origin,
where the former includes polysaccharides, proteins, glycoproteins,
cell-wall fragments etc. derived from fungi, bacteria and even plants
and the latter includes metal ions, UV light, chemically defined
compounds and any other stresses which are of non-biological origin.
The success of elicitation as an effective strategy for improving second-
ary metabolite production in plant cell cultures has prompted many
reviews (Namdeo, 2007; Radman et al., 2003) and studies on the mech-
anisms of elicitor action (Vasconsuelo and Boland, 2007; Zhao et al.,
2005a). Though the strategy has been used predominantly in plant sys-
tems, application of elicitation to microbial systems is also known and is
becoming increasingly popular, especially with endophytic fungal
systems where host plant signaling plays a very important role in
secondary metabolism. Interestingly, in the case of endophytic fungi,
even small molecules like ethanol have caused significant enhancement
in the product yield (Venugopalan and Srivastava, 2015; Zhao et al.,
2013c), which supports the idea that elicitation can become a cost-
effective, yield and productivity enhancement strategy for sustainable
metabolite production in endophytic fungal fermentations at large
scale. Further examples related to the application of elicitation strategy
for improved product yields in endophytic fungi have been listed in
Table 5.
In addition to elicitors, use of metabolic inhibitors in order to divert
the flux toward the product of interest has also been documented.
Specifically, in the case of a taxol producing endophyte, which also pro-
duced large amounts of ergosterol, use of sterol biosynthesis inhibitors
to divert the geranyl-geranyl pyrophosphate pool toward taxol biosyn-
thesis resulted in the enhancement of the taxol yield up to 50 fold
(Li et al., 1998b). Examples of inhibitor addition for improved product
yields in endophytic fungi are listed in Table 6.
5.2.3. Precursor feeding
Precursor feeding is the strategy of exogenously supplying a biosyn-
thetic precursor or other intermediates in the biosynthetic pathway to
the culture medium in order to increase the desired product yield
(Mulabagal and Tsay, 2004). It is a widely used strategy in plant cell/
tissue cultivation and is now being explored in the fermentation of
endophytic fungi also. It is feasible in cases where the precursors or in-
termediates are inexpensive. While a high concentration of precursors
may usually increase reaction rates, the toxicity to cells may also
increase with increase in concentration, and so the concentration of pre-
cursor to be added to an endophytic fungal culture has to be optimized
carefully (Gaosheng and Jingming, 2012). Precursor feeding studies
have also played a major part in the elucidation of biosynthetic
pathways of various metabolites including taxol and camptothecin
(Guerra-Bubb et al., 2012; Yamazaki et al., 2004). Stierle et al. (1993)
used isotope-labeled precursors to establish the existence of taxol
biosynthetic machinery in the fungal culture, T. andreanae. Feeding of
the precursor leucine to the camptothecin producing Entrophospora
infrequens was used to establish that leucine rather than mevalonic
acid is the precursor for the terpenoid moiety of the camptothecin mol-
ecule (Amna et al., 2012). Examples related to the effect of precursor
feeding on the product yield in endophytic fungi have been listed in
Table 7.
5.2.4. Use of adsorbent resins/solid supports
In industrial processes, downstream processing is easier if the
fermentation product is extracellular rather than being stored inside
the cell. Further, in the case of self-toxic metabolites immediate removal

Table 6
Examples of inhibitor addition for improvement in product yield from endophytes.

Product Organism Inhibitor Effect Reference

Taxol Pestalotiopsis microspora Ribonuclease inhibitors (vanadyl sulfate, lauryl sulfate, bentonite, and polyvinyl sulfate) Positive (vanadyl sulfate) Li et al. (1998b)
Ne32 Sterol biosynthesis inhibitors (paclobutrazol, tebuconazole, triadimenol, lovastatin, triadimefon, 50 fold yield increase (triadimefon)
2-chloroethylphosphonic acid, arachidonic acid, 1-aminobenzotriazole, fluconazole)

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10 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx

Table 7
Examples of precursor feeding for improvement in product yield from endophytes.

Product Organism Precursor Effect Reference

Taxol Pestalotiopsis microspora Ne32 Sodium benzoate Positive Li et al. (1998b)

Camptothecin Trichoderma atroviride LY357 Tryptamine Positive Pu et al. (2013)
Secologanin Negative
Camptothecin Entrophospora infrequens RJMEF001 Tryptophan Positive Amna et al. (2012)
Tryptamine Positive
Geraniol No camptothecin
Citral No camptothecin
Mevalonic acid + tryptophan Negative
Leucine + tryptophan Positive
Geraniol + tryptophan No camptothecin
Citral + tryptophan No camptothecin

of the product is necessary to sustain higher yields, as the growth of the
organism is affected by the release of its toxic metabolites (Singh et al.,
2010). In situ product removal (ISPR) is an integrated bioprocess
involving simultaneous production and separation. It confers several
advantages on the fermentation process including improved product
recovery, elimination of feedback inhibition, overcoming product deg-
radation, avoiding product auto-toxicity, reducing cost and improving
the product yield efficiently (Luo et al., 2014; Schügerl and Hubbuch,
2005). Two ways of employing ISPR are extraction with a liquid solvent
or adsorption with a solid sorbent added to the culture medium (Xu
et al., 2009). The latter strategy, which has the advantage of lower risk
of toxicity as inert polymeric resins are employed as opposed to reactive
organic solvents, has been employed extensively in endophytic fungal
fermentation (Table 8). Several parameters like the type, concentration,
time of addition and incubation period of resin need to be selected and
optimized for improving metabolite production in the suspension
culture of the endophytes. Recently, addition of adsorbent resins
has been explored as a potential strategy to trigger the differential
expression of secondary metabolites in fungal strains, which could
lead to the discovery of novel bioactive molecules (González-
Menéndez et al., 2014).
Another strategy of yield enhancement or even novel metabolite
discovery is the use of inert solid supports like polypropylene, cellulose,
absorbent cotton gauze, polyester–cellulose, or polyurethane in solid-
state and submerged fermentation (Bigelis et al., 2006). Culturing
fungi on static supports in liquid medium enable exploitation of various
environmental stimuli that influence metabolism, morphogenesis, and
differentiation thus affecting their metabolite profile. The presence of
the solid support may mimic natural fungal habitats thus triggering
changes in the growth and physiology of the culture (Bigelis et al.,
2006). The physicochemical characteristics of the support system and
its interaction with available water, oxygen, and medium components
and other factors like availability of the gaseous phase in interstitial
spaces and aeration, nutrient and waste product absorbance and
diffusion, induce environmental conditions which mimic those found
in nature. This strategy has been successfully applied to the endophytic
strain of Phomopsis sp. using absorbent cotton gauze and cellulose
paper for yield enhancement of mycoepoxydiene by 1 and 1.2 fold,
respectively (Thammajaruk et al., 2011).
6. Strategies to unlock the cryptic/silenced gene clusters
in endophytes
While the application of bioprocess optimization strategies on
endophytic fungi in axenic culture has led to increased product yields
in many cases, most often genomic instability or silencing of the
biosynthetic genes in axenic culture hinders attempts of bioprocess
development using newly isolated strains. Hence, the challenges associ-
ated with stabilizing the productive capabilities of the axenic culture of
endophytes can be addressed via the use of strategies like co-cultivation
and epigenetic modifier addition. A rational approach toward exploiting
the axenic cultures of endophytes for secondary metabolite production
has been depicted in Fig. 2.
6.1. Co-cultivation and mixed fermentation
Currently, studies with respect to bioprospecting of endophytes and
exploitation of endophytes for known high value metabolites have been
done using the classical approach of screening several different endo-
phytes from a host plant and identifying competent endophytes with
the desired trait i.e. the ability to produce a specific metabolite while
the remaining strains are discarded without further explorations
(Kusari et al., 2012a). Further, the identified endophytes are maintained
as axenic monocultures for fermentation of the desired product. On one
hand, in axenic culture, the endophyte no longer has access to the spe-
cialized microenvironment of the host plant which it is attuned to,
which may lead to silencing of its secondary metabolite genes, and on
the other hand standard culture conditions may not be sufficient to
trigger expression of the cryptic biosynthetic gene clusters present in
fungal endophytes leading to a redundancy in terms of novel metabolite
discovery. An important finding via the use of whole-genome sequenc-
ing strategies is that the number of genes encoding the biosynthetic
enzymes in various fungi and bacteria is greater than the known sec-
ondary metabolites of these microbes (Scherlach and Hertweck,
2009). Slight changes in culture parameters are known to significantly
affect the metabolic processes in microbes, especially in the case of en-
dophytes whose survival is defined by a delicately balanced relationship
with the host plant featuring complex multipartite interactions with
other endophytes. Examples exist to indicate the sensitivity of the

Table 8
Use of adsorbent resins for improvement in product yield from endophytes.

Product Organism Resin type Yield enhancement Reference

Beauvericin Fusarium redolens Dzf2 Polystyrene resin (X-5) 1.8 fold Xu et al. (2009)
Palmarumycin C13 Berkleasmium sp. Dzf12 Polystyrene resin (AB-8) 1.4 fold Zhao et al. (2011a)
Botrallin Hyalodendriella sp. ponipodef12 Polystyrene resin DM-301 2.29 fold Luo et al. (2014)
TMC-264 Hyalodendriella sp. ponipodef12 Polystyrene resin DM-301 11.76 fold Luo et al. (2014)
Camptothecin Trichoderma atroviride LY357 HP20 11 fold Pu et al. (2013)
XAD16 4.4 fold

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx 11

Fig. 2. A rational approach toward the exploitation of axenic cultures of endophytes for secondary metabolite production.

metabolic machinery that endophytes exhibit to the slightest changes
in culture conditions. Paranagama et al. (2007) reported the production
of six new secondary metabolites by the endophyte, Paraphaeosphaeria
quadriseptata when distilled water was used in the medium in
place of tap water. Similarly, when the medium was changed from
solid to liquid, Chaetomium chiversii produced radicicol instead of
chaetochromin A.
Apart from screening of individual culture parameters, co-
cultivation or mixed fermentation is another OSMAC strategy that can
enable simulation of the natural habitat of endophytes. Designing
novel co-culture systems which mimic the host microenvironment by
providing the endophyte with the same kind of in planta selection
pressure under which it thrives can prove to be beneficial with
respect to host-associated and novel secondary metabolite yields in
endophytes. This requires a complete understanding of the complex
ecological interactions between endophytes and plants and also
among the various endophytes that co-exist within the same host.
Co-cultivation/mixed fermentation strategy has been employed and re-
ported for novel product isolation and as a yield enhancement strategy
(Table 9).

Table 9
Use of co-cultivation/mixed fermentation strategy for improvement in product yield and novel product discovery from endophytes.

Compound Co-culture/mixed fermentation system Outcome Reference

Taxol Paraconiothyrium strain Alternaria sp. 2.7 fold yield increase Soliman and Raizada
SSM001 Phomopsis sp. 3.8 fold yield increase (2013)
Alternaria sp. and Phomopsis sp. 7.8 fold yield increase
Marinamide and marinamide methylether Two unidentified mangrove-derived endophytic fungi Novel alkaloids with potent cytotoxicity Zhu and Lin (2006)
6-Methylsalicylic acid Two unidentified mangrove-derived endophytic fungi Induced only in co-culture Zhu et al. (2007)
Cyclo-(phe–phe) dipeptide
Subenniatins A and B Fusarium tricinctum Fusarium begonia Novel linear depsipeptides Wang et al. (2013)
Acremostatins A, B and C Acremonium sp. Mycogone rosea Novel lipoaminopeptide Degenkolb et al. (2002)
Lateropyrone F. tricinctum Bacillus subtilis 1.4 fold increase Ola et al. (2013)
Enniatin A1 78 fold increase
Enniatin B 9 fold increase
Enniatin B1 36.3 fold increase
Fusaristatin A 7.9 fold increase
Macrocarpon C Novel substituted pyranone
N-(carboxymethyl)-anthranilic acid Novel starting material for indigo synthesis
(−)-Citreoisocoumarin Novel metabolite
(−)-Citreoisocoumarinol Novel metabolite
Enniatins A1, B1, and B (cyclic depsipeptides) F. tricinctum Streptomyces lividans Induced only in co-culture Ola et al. (2013)
Fusaristatin A — lipopeptide Induced only in co-culture
Red resin (traditional Chinese medicine) Dracaena cochinchinensis Fusarium moniliforme 9568D Resin accumulation in inoculated sites Jiang et al. (2003)
Taxol Taxus chinensis var. maireiFusarium mairei 38 fold yield increase Li et al. (2009)
Taxol Taxus cuspidata F. mairei 4.3 fold yield increase Li and Tao (2009)
Podophyllotoxin Linum album Piriformospora indicaa 3.4 fold yield increase Baldi et al. (2010)
Sebacina vermiferaa 3.8 fold yield increase
6-Methoxy podophyllotoxin L. album P. indicaa 4.9 fold yield increase Baldi et al. (2010)
S. vermiferaa 8.7 fold yield increase
Stemphyperylenol (anti-fungal polyketide) Alternaria tenuissima Nigrospora sphaerica Induced only in co-culture Chagas et al. (2013)
13-Oxo-9,11-octadecadienoic acid (oxylipin) Endophyte Paraconiothyrium Phytopathogen Fusarium Decreased the mycotoxin production by Combès et al. (2012)
variabile oxysporum F. oxysporum
a
Arbuscular mycorrhizae-like fungi.

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
12 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx

Table 10
Use of epigenetic modifiers for novel product discovery from endophytes.

Epigenetic modifier used Organism Effect Reference

Suberoylanilide hydroxamic acid (SBHA) Endophytic Fusarium Production of novel fusaric acid derivative: Chen et al. (2013)
(HDAC inhibitor) oxysporum strain R1 • 5-Butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid
• 5-(But-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid
Sodium butyrate (HDAC inhibitor) Leucostoma persoonii • Novel compound — cytosporone R Beau et al. (2012)
• Enhanced production of known cytosporones B (360%), C (580%) and E (890%)
5-Azacytidine (DNMT inhibitor) and/or Endophytic Alternaria sp. Production of mycotoxins including alternariol, alternariol-5-O-methyl ether, Sun et al. (2012)
suberoylanilide hydroxamic acid 3′-hydroxyalternariol-5-O-methyl ether, altenusin, tenuazonic acid, and altertoxin II.
(HDAC inhibitor)
Suberoylanilide hydroxamic acid (HDAC Pestalotiopsis acacia Production of 3 novel aromatic Yang et al. (2013)
inhibitor) + 5-azacytidine Compounds:
(DNMT inhibitor) • 20-Hydroxy-60-hydroxymethyl-40-methylphenyl-2,6-dihydroxy-3-(2-isopentenyl)benzoate
• 4,6-Dihydroxy-7-hydroxymethyl-3-methylcoumarin
• 4,6-dihydroxy-3,7-dimethylcoumarin and five known polyketides:
Endocrocin
Pestalotiollide B
Pestalotiopyrone G
Scirpyrone A
7-Hydroxy-2-(2-hydroxypropyl)-5-methylchromone
5-Azacytidine (DNMT inhibitor) Pestalotiopsis crassiuscula Production of 7 metabolites: Yang et al. (2014b)
• 4,6-Dihydroxy-7-hydroxymethyl-3-methoxymethylcoumarin (novel metabolite)
• 4,6-Dihydroxy-7-hydroxymethyl-3-methylcoumarin
• 4,6-Dihydroxy-3,7-dimethylcoumarin
• Pestalotiopyrone G
• Scirpyrone A
• Scirpyrone B
• 2′-Hydroxy-6′-hydroxymethyl-4′-methylphenyl 2,6-dihydroxy-3-(2-isopentenyl) benzoate
Nicotinamide (an NAD+-dependent Graphiopsis chlorocephala Production of 6 novel benzophenones: cephalanones A–F and a known metabolite: Asai et al. (2013)
HDAC inhibitor) 2-(2,6-dihydroxy-4-methylbenzoyl)-6-hydroxybenzoic acid

Designing a successful co-cultivation system involves several
challenges. An optimum inoculum ratio of the various species being
co-cultured should be established such that the competition between
the species is balanced and the growth of one does not overpower
the other. Culture conditions and medium composition have to be opti-
mized to accommodate the different growth morphologies being used
in the co-culture system, while also maximizing productivity of the
desired product by one of the organisms. Reactor design is also an im-
portant factor to be considered (Weathers et al., 2010). When these
challenges are addressed, co-cultivation/mixed fermentation becomes
a powerful strategy as the competition or antagonism experienced
during co-cultivation may lead to a significantly enhanced production
of constitutively present compounds and/or to an accumulation of
cryptic compounds that are not detected in axenic culture of the select-
ed strain (Marmann et al., 2014).
6.2. Use of epigenetic modifiers
Fungal biosynthetic gene clusters, which are often located in the
distal regions of chromosomes, exist in a heterochromatin state. These
genes are often transcriptionally controlled by epigenetic regulation
such as histone deacetylation and DNA methylation (Pettit, 2010).
Histone deacetylation and DNA methylation operate to modify the chro-
matin and thus regulate gene expression or silencing in fungi and other
organisms. Hence, the possibility of using epigenetic modifiers like DNA
methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors
to induce chromatin remodeling has been proposed as a strategy for
unlocking cryptic biosynthetic gene clusters (Brakhage and Schroeckh,
2011). Commonly used DNMT inhibitors include 5-azacytidine and
5-aza-20-deoxycytidine, while HDAC inhibitors include hydroxamic-
acid containing compounds or cyclic peptides such as trichostatin A
and trapoxin B (Cichewicz, 2010). Table 10 lists the examples available
on the usage of epigenetic modifiers for novel product discovery in
endophytes.
There are many literature reviews available on the regulatory
elements that modulate the transcription of secondary metabolite
genes (Brakhage, 2013; Keller et al., 2005). An understanding of the
regulation of secondary metabolite synthesis has resulted in several
novel approaches for the isolation of new secondary metabolites from
filamentous fungi. The proposed strategies include over-expression of
a cluster-specific transcription factor (TF) gene, exchange of promoters
for biosynthesis genes or TF genes with inducible promoters such as the
alcohol dehydrogenase A gene promoter, and over-expression or
deletion of a global regulator gene (Brakhage, 2013; Scharf and
Brakhage, 2013). These methods can be applied to endophytic fungi as
well to modulate their secondary metabolite machinery.
7. Conclusions
The vast amount of knowledge that is already available and the
newer pieces of the puzzle that are emerging in endophyte research
only make it obvious that endophytic fungi will be one of the major
sources of secondary metabolites in the near future. In addition, consid-
ering the various yield enhancement strategies available that can be
tailored to endophytic fungi, the future potential of endophytic fungi
research for the production of high value natural products looks
extremely promising. However, a much greater understanding of the
host plant-endophyte dynamics at the molecular level is essential to
exploit the promising potential of these extraordinary organisms at
commercial level. This will also enable rational optimization of the
bioprocess for high and sustainable production of high value secondary
metabolites from endophytes.
References
Adrio, J.L., Demain, A.L., 2006. Genetic improvement of processes yielding microbial prod-
ucts. FEMS Microbiol. Rev. 30, 187–214.
Ajikumar, P.K., Xiao, W.H., Tyo, K.E.J., Wang, Y., Simeon, F., Leonard, E., et al., 2010. Iso-
prenoid pathway optimization for taxol precursor overproduction in Escherichia
coli. Science 330, 70–74.
Alam, A., Naik, P.K., Mishra, G.P., 2009. Congruence of RAPD and ISSR markers for evalua-
tion of genomic relationship among 28 populations of Podophyllum hexandrum Royle
from Himachal Pradesh, India. Turk. J. Bot. 33, 1–12.
Amna, T., Amina, M., Sharma, P.R., Puri, S.C., Al-Youssef, H.M., Al-Taweel, A.M., et al., 2012.
Effect of precursors feeding and media manipulation on production of novel antican-
cer pro-drug camptothecin from endophytic fungus. Braz. J. Microbiol. 43, 1476–1489.

Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
Arnold, A.E., Lewis, L.C., 2005. Ecology and evolution of fungal endophytes, and their roles
against insects. In: Vega, F., Blackwell, M. (Eds.), Insect–Fungal Associations: Ecology
and Evolution. Oxford University Press, New York, pp. 74–96.
Arnold, A.E., Mejía, L.C., Kyllo, D., Rojas, E.I., Maynard, Z., Robbins, N., et al., 2003. Fungal
endophytes limit pathogen damage in a tropical tree. Proc. Natl. Acad. Sci. 100,
15649–15654.
Asai, T., Otsuki, S., Sakurai, H., Yamashita, K., Ozeki, T., Oshima, Y., 2013. Benzophenones
from an endophytic fungus, Graphiopsis chlorocephala, from Paeonia lactiflora cultivat-
ed in the presence of an NAD+-dependent HDAC inhibitor. Org. Lett. 15, 2058–2061.
Baldi, A., Srivastava, A.K., Bisaria, V.S., 2009. Fungal elicitors for enhanced production of
secondary metabolites in plant cell suspension cultures. In: Varma, A., Kharkwal,
A.C. (Eds.), Symbiotic Fungi. Springer, Berlin Heidelberg, pp. 373–380.
Baldi, A., Farkya, S., Jain, A., Gupta, N., Mehra, R., Datta, V., et al., 2010. Enhanced produc-
tion of podophyllotoxins by co-culture of transformed Linum album cells with plant
growth-promoting fungi. Pure Appl. Chem. 82, 227–241.
Barrios-Gonzalez, J., Fernandez, F.J., Tomasini, A., 2003. Microbial secondary metabolites
production and strain improvement. Indian J. Biotechnol. 2, 322–333.
Beau, J., Mahid, N., Burda, W.N., Harrington, L., Shaw, L.N., Mutka, T., et al., 2012. Epigenet-
ic tailoring for the production of anti-infective cytosporones from the marine fungus
Leucostoma persoonii. Mar. Drugs 10, 762–774.
Bigelis, R., He, H., Yang, H.Y., Chang, L.P., Greenstein, M., 2006. Production of fungal
antibiotics using polymeric solid supports in solid-state and liquid fermentation.
J. Ind. Microbiol. Biotechnol. 33, 815–826.
Bode, H.B., Bethe, B., Hӧfs, R., Zeeck, A., 2002. Big effects from small changes: possible
ways to explore nature's chemical diversity. Chembiochem 3, 619–627.
Bӧlker, M., Bӧhnert, H.U., Braun, K.H., Gӧrl, J., Kahmann, R., 1995. Tagging pathogenicity
genes in Ustilago maydis by restriction enzyme-mediated integration (REMI). Mol.
Gen. Genet. 248, 547–552.
Brakhage, A.A., 2013. Regulation of fungal secondary metabolism. Nat. Rev. Microbiol. 11,
21–32.
Brakhage, A.A., Schroeckh, V., 2011. Fungal secondary metabolites—strategies to activate
silent gene clusters. Fungal Genet. Biol. 48, 15–22.
Brundrett, M., 2004. Diversity and classification of mycorrhizal associations. Biol. Rev. 79,
473–495.
Carroll, G.C., 1991. Beyond pest deterrence — alternative strategies and hidden costs of
endophytic mutualisms in vascular plants. In: Andrews, J.A., Hirano, S.S. (Eds.), Micro-
bial Ecology of Leaves. Springer-Verlag, New York, pp. 358–375.
Cassady, J.M., Chan, K.K., Floss, H.G., Leistner, E., 2004. Recent developments in the
maytansinoid antitumor agents. Chem. Pharm. Bull. 52, 1–26.
Chagas, F.O., Dias, L.G., Pupo, M.T., 2013. A mixed culture of endophytic fungi increases
production of antifungal polyketides. J. Chem. Ecol. 39, 1335–1342.
Chaichanan, J., Wiyakrutta, S., Pongtharangkul, T., Isarangkul, D., Meevootisom, V., 2014.
Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra
L38. Braz. J. Microbiol. 45, 287–293.
Chattopadhyay, S., Farkya, S., Srivastava, A.K., Bisaria, V.S., 2002. Bioprocess considerations
for production of secondary metabolites by plant cell suspension cultures. Biotechnol.
Bioprocess Eng. 7, 138–149.
Chaturvedi, P., Gajbhiye, S., Roy, S., Dudhale, R., Chowdhary, A., 2014. Determination of
kaempferol in extracts of Fusarium chlamydosporum, an endophytic fungi of
Tylophora indica (Asclepeadaceae) and its anti-microbial activity. IOSR J. Pharm.
Biol. Sci. 9, 51–55.
Chen, H.J., Awakawa, T., Sun, J.Y., Wakimoto, T., Abe, I., 2013. Epigenetic modifier-induced
biosynthesis of novel fusaric acid derivatives in endophytic fungi from Datura stramo-
nium L. Nat. Prod. Bioprospect. 3, 20–23.
Cichewicz, R.H., 2010. Epigenome manipulation as a pathway to new natural product
scaffolds and their congeners. Nat. Prod. Rep. 27, 11–22.
Combès, A., Ndoye, I., Bance, C., Bruzaud, J., Djediat, C., Dupont, J., et al., 2012. Chemical
communication between the endophytic fungus Paraconiothyrium variabile and the
phytopathogen Fusarium oxysporum. PLoS ONE 7, e47313.
Cui, Y., Yi, D., Bai, X., Sun, B., Zhao, Y., Zhang, Y., 2012. Ginkgolide B produced endophytic
fungus (Fusarium oxysporum) isolated from Ginkgo biloba. Fitoterapia 83, 913–920.
Dai, W.L., Tao, W.Y., 2008. Preliminary study on fermentation conditions of taxol-
producing endophytic fungus. Chem. Ind. Eng. Prog. 27, 883–886.
Dangi, R., Misar, A., Tamhankar, S., Rao, S., 2014. Diosgenin content in some Trigonella
species. Indian J. Adv. Plant Res. 1, 47–51.
Degenkolb, T., Heinze, S., Schlegel, B., Strobel, G., Gräfe, U., 2002. Formation of new
lipoaminopeptides, acremostatins A, B, and C, by co-cultivation of Acremonium sp.
Tbp-5 and Mycogone rosea DSM 12973. Biosci. Biotechnol. Biochem. 66, 883–886.
Dejong, J.M., Liu, Y., Bollon, A.P., Long, R.M., Jennewein, S., Williams, D., et al., 2006.
Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae.
Biotechnol. Bioeng. 93, 212–224.
Dubey, D., Rath, S., Sahu, M.C., Debata, N.K., Padhy, R.N., 2012. Antimicrobials of plant or-
igin against TB and other infections and economics of plant drugs-introspection.
Indian J. Tradit. Knowl. 11, 225–233.
Flores-Bustamante, Z.R., Rivera-Orduña, F.N., Martínez-Cárdenas, A., Flores-Cotera, L.B.,
2010. Microbial paclitaxel: advances and perspectives. J. Antibiot. (Tokyo) 63,
460–467.
Gao, Y., Yin, H., Sun, Y., Zhang, Z., Cui, Y., 2008. Mutagenesis of a berberine-producing en-
dophytic fungus. J. Fungal Res. 4, 6.
Gaosheng, H., Jingming, J., 2012. Production of useful secondary metabolites through reg-
ulation of biosynthetic pathway in cell and tissue suspension culture of medicinal
plants. In: Leva, A., Rinalsi, L.M.R. (Eds.), Recent Advances in Plant in Vitro Culture.
InTech, pp. 197–210.
González-Menéndez, V., Asensio, F., Moreno, C., de Pedro, N., Monteiro, M.C., de la Cruz,
M., et al., 2014. Assessing the effects of adsorptive polymeric resin additions on fungal
secondary metabolite chemical diversity. Mycology 5, 179–191.
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
13
Grond, S., Papastavrou, I., Zeeck, A., 2002. Novel α-L-rhamnopyranosides from a single
strain of Streptomyces by supplement-induced biosynthetic steps. Eur. J. Org. Chem.
2002, 3237–3242.
Guerra-Bubb, J., Croteau, R., Williams, R.M., 2012. The early stages of taxol biosynthesis:
an interim report on the synthesis and identification of early pathway metabolites.
Nat. Prod. Rep. 29, 683–696.
Hedden, P., Phillips, A.L., Rojas, M.C., Carrera, E., Tudzynski, B., 2001. Gibberellin biosyn-
thesis in plants and fungi: a case of convergent evolution? J. Plant Growth Regul.
20, 319–331.
Heinig, U., Scholz, S., Jennewein, S., 2013. Getting to the bottom of Taxol biosynthesis by
fungi. Fungal Divers. 60, 161–170.
Hendrawati, O., Woerdenbag, H.J., Hille, J., Kayser, O., 2012. Metabolic engineering of me-
dicinal plants and microorganisms for the production of natural products. In: Kayser,
O., Warzecha, H. (Eds.), Pharmaceutical Biotechnology: Drug Discovery and Clinical
Applications, Second Edition Wiley‐VCH Verlag GmbH & Co. KGaA, pp. 491–526.
Hong, Y.I.N., Yang, G.A.O., 2010. Optimization of fermentation conditions of a
mutagenized berberine-producing strain. J. Fungal Res. 1, 15.
Howitz, K.T., Sinclair, D.A., 2008. Xenohormesis: sensing the chemical cues of other
species. Cell 387–391.
Hyde, K.D., Soytong, K., 2008. The fungal endophyte dilemma. Fungal Divers. 33, 163–173.
Jiang, D.F., Ma, P., Yang, J., Wang, X., Xu, K., Huang, Y., et al., 2003. Formation of blood resin
in abiotic Dracaena cochinchinensis inoculated with Fusarium 9568D. Ying Yong Sheng
Tai Xue Bao 14, 477–478.
Kai, Z., Xuan, W., Yushi, S., Ying, W., Wenxiang, P., Dongpo, Z., 2008. Screening of high
taxol producing fungi by NTG combining with UV mutagenesis. J. Nat. Sci. Heilong-
jiang Univ. 1, 16.
Kai, Z., Qingshen, S., Yanjun, Z., Wenxiang, P., Tao, J., Dongpo, Z., 2009. Screening and
characterization of a high taxol producing fungus by protoplast mutagenesis. High
Technol. Lett. 15, 220–226.
Karuppusamy, S., 2009. A review on trends in production of secondary metabolites from
higher plants by in vitro tissue, organ and cell cultures. J. Med. Plants Res. 3,
1222–1239.
Kaul, S., Ahmed, M., Zargar, K., Sharma, P., Dhar, M.K., 2012. Prospecting endophytic
fungal assemblage of Digitalis lanata Ehrh. (foxglove) as a novel source of digoxin:
a cardiac glycoside. Biotech 3, 335–340.
Keller, N.P., Turner, G., Bennett, J.W., 2005. Fungal secondary metabolism—from biochem-
istry to genomics. Nat. Rev. Microbiol. 3, 937–947.
Kobayashi, D.Y., Palumbo, J.D., 2000. Bacterial endophytes and their effects on plants and
uses in agriculture. In: Bacon, C.W., White, J.F. (Eds.), Microbial Endophytes. Marcel
Dekker, New York, pp. 199–236.
Krings, M., Taylor, T.N., Hass, H., Kerp, H., Dotzler, N., Hermsen, E.J., 2007. Fungal endo-
phytes in a 400-million-yr-old land plant: infection pathways, spatial distribution,
and host responses. New Phytol. 174, 648–657.
Kumara, P.M., Zuehlke, S., Priti, V., Ramesha, B.T., Shweta, S., Ravikanth, G., et al., 2012.
Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.
f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie
Van Leeuwenhoek 101, 323–329.
Kurland, C.G., Canback, B., Berg, O.G., 2003. Horizontal gene transfer: a critical view. Proc.
Natl. Acad. Sci. 100, 9658–9662.
Kusari, S., Košuth, J., Čellárová, E., Spiteller, M., 2011a. Survival-strategies of endophytic
Fusarium solani against indigenous camptothecin biosynthesis. Fungal Ecol. 4,
219–223.
Kusari, S., Zühlke, S., Spiteller, M., 2011b. Effect of artificial reconstitution of the interac-
tion between the plant Camptotheca acuminata and the fungal endophyte Fusarium
solani on camptothecin biosynthesis. J. Nat. Prod. 74, 764–775.
Kusari, S., Hertweck, C., Spiteller, M., 2012a. Chemical ecology of endophytic fungi: origins
of secondary metabolites. Chem. Biol. 19, 792–798.
Kusari, S., Verma, V.C., Lamshoeft, M., Spiteller, M., 2012b. An endophytic fungus from
Azadirachta indica A. Juss. that produces azadirachtin. World J. Microbiol. Biotechnol.
28, 1287–1294.
Kusari, S., Singh, S., Jayabaskaran, C., 2014. Rethinking production of Taxol (paclitaxel)
using endophyte biotechnology. Trends Biotechnol. 32, 304–311.
Lee, J.C., Lobkovsky, E., Pliam, N.B., Strobel, G., Clardy, J., 1995. Subglutinols A and B: im-
munosuppressive compounds from the endophytic fungus Fusarium subglutinans.
J. Org. Chem. 60, 7076–7077.
Li, Y.C., Tao, W.Y., 2009. Interactions of Taxol-producing endophytic fungus with its host
Taxus spp. during Taxol accumulation. Cell Biol. Int. 33, 106–112.
Li, J.Y., Sidhu, R.S., Ford, E., Hess, W.M., Strobel, G.A., 1998a. The induction of taxol
production in the endophytic fungus — Periconia sp. from Torreya grandifolia. J. Ind.
Microbiol. 20, 259–264.
Li, J.Y., Sidhu, R.S., Bollon, A., Strobel, G.A., 1998b. Stimulation of taxol production in liquid
cultures of Pestalotiopsis microspora. Mycol. Res. 102, 461–464.
Li, Y.C., Tao, W.Y., Cheng, L., 2009. Paclitaxel production using co-culture of Taxus
suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor. Appl.
Microbiol. Biotechnol. 83, 233–239.
Li, P., Xu, L., Mou, Y., Shan, T., Mao, Z., Lu, S., et al., 2012a. Medium optimization for
exopolysaccharide production in liquid culture of endophytic fungus Berkleasmium
sp. Dzf12. Int. J. Mol. Sci. 13, 11411–11426.
Li, Y., Li, P., Mou, Y., Zhao, J., Shan, T., Ding, C., et al., 2012b. Enhancement of diepoxin ζ
production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 by polysac-
charides from its host plant Dioscorea zingiberensis. World J. Microbiol. Biotechnol. 28,
1407–1413.
Li, Y., Shan, T., Mou, Y., Li, P., Zhao, J., Zhao, W., et al., 2012c. Enhancement of
palmarumycin C12 and C13 production in liquid culture of the endophytic fungus
Berkleasmium sp. Dzf12 by oligosaccharides from its host plant Dioscorea
zingiberensis. Molecules 17, 3761–3773.
14 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
Linden, J.C., 2006. Secondary products from plant tissue culture. Encycl. Life Support Syst.
(UNESCO-EOLSS) Biotechnol. 4, 1–9.
Liu, K., Ding, X., Deng, B., Chen, W., 2010. 10-Hydroxycamptothecin produced by a new
endophytic Xylaria sp., M20, from Camptotheca acuminata. Biotechnol. Lett. 32,
689–693.
Liu, L., Wei, Y.M., Zhou, X.W., Lin, J., Sun, X.F., Tang, K.X., 2013. Agrobacterium tumefaciens-
mediated genetic transformation of the Taxol-producing endophytic fungus Ozonium
sp EFY21. Genet. Mol. Res. 12, 2913.
Long, D.M., Smidmansky, E.D., Archer, A.J., Strobel, G.A., 1998. In vivo addition of telomeric
repeats to foreign DNA generates chromosomal DNAs in the taxol-producing fungus
Pestalotiopsis microspora. Fungal Genet. Biol. 24, 335–344.
Luo, H., Liu, H., Cao, Y., Xu, D., Mao, Z., Mou, Y., et al., 2014. Enhanced production of
botrallin and TMC-264 with in situ macroporous resin adsorption in mycelial liquid
culture of the endophytic fungus Hyalodendriella sp. Ponipodef12. Molecules 19,
14221–14234.
Ma, C., Jiang, D., Wei, X., 2011. Mutation breeding of Emericella foeniculicola TR21 for im-
proved production of tanshinone IIA. Process Biochem. 46, 2059–2063.
Malik, S., Cusidó, R.M., Mirjalili, M.H., Moyano, E., Palazón, J., Bonfill, M., 2011. Production
of the anticancer drug taxol in Taxus baccata suspension cultures: a review. Process
Biochem. 46, 23–34.
Mangathayaru, K., 2013. Pharmacognosy: An Indian perspective. Pearson Education,
India.
Manivasakam, P., Aubrecht, J., Sidhom, S., Schiestl, R.H., 2001. Restriction enzymes in-
crease efficiencies of illegitimate DNA integration but decrease homologous integra-
tion in mammalian cells. Nucleic Acids Res. 29, 4826–4833.
Marler, M., Pedersen, D., Mitchell-Olds, T., Callaway, R.M., 1999. A polymerase chain
reaction method for detecting dwarf mistletoe infection in Douglas-fir and western
larch. Can. J. For. Res. 29, 1317–1321.
Marmann, A., Aly, A.H., Lin, W., Wang, B., Proksch, P., 2014. Co-cultivation—a powerful
emerging tool for enhancing the chemical diversity of microorganisms. Mar. Drugs
12, 1043–1065.
Medema, M.H., Trefzer, A., Kovalchuk, A., van den Berg, M., Müller, U., Heijne, W., et al.,
2010. The sequence of a 1.8-Mb bacterial linear plasmid reveals a rich evolutionary
reservoir of secondary metabolic pathways. Genome Biol. Evol. 2, 212–224.
Ming, Q., Han, T., Li, W., Zhang, Q., Zhang, H., Zheng, C., et al., 2012. Tanshinone IIA and
tanshinone I production by Trichoderma atroviride D16, an endophytic fungus in
Salvia miltiorrhiza. Phytomedicine 19, 330–333.
Mochizuki, S., Hiratsu, K., Suwa, M., Ishii, T., Sugino, F., Yamada, K., et al., 2003. The large
linear plasmid pSLA2‐L of Streptomyces rochei has an unusually condensed gene
organization for secondary metabolism. Mol. Microbiol. 48, 1501–1510.
Mou, Y., Luo, H., Mao, Z., Shan, T., Sun, W., Zhou, K., et al., 2013. Enhancement of
palmarumycins C12 and C13 production in liquid culture of endophytic fungus
Berkleasmium sp. Dzf12 after treatments with metal ions. Int. J. Mol. Sci. 14,
979–998.
Mu, J.H., Bollon, A.P., Sidhu, R.S., 1999. Analysis of β-tubulin cDNAs from taxol-resistant
Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of
the taxol-binding properties of their products. Mol. Gen. Genet. 262, 857–868.
Mulabagal, V., Tsay, H.S., 2004. Plant cell cultures-an alternative and efficient source for
the production of biologically important secondary metabolites. Int. J. Appl. Sci. Eng.
2, 29–48.
Musavi, S.F., Dhavale, A., Balakrishnan, R.M., 2014. Optimization and kinetic modelling of
cell associated camptothecin production from an endophytic Fusarium oxysporum
NFX06. Prep. Biochem. Biotechnol. 45, 158–172.
Nair, D.N., Padmavathy, S., 2014. Impact of endophytic microorganisms on plants,
environment and humans. Sci. World J. 2014, 1–11.
Namdeo, A.G., 2007. Plant cell elicitation for production of secondary metabolites: a
review. Pharmacogn. Rev. 1, 69–79.
Newman, D.J., Cragg, G.M., 2007. Natural products as sources of new drugs over the last
25 years. J. Nat. Prod. 70, 461–477.
Nirupama, R., Chaithra, K., Govindappa, M., Chandrappa, C.P., Vinay, R.B., 2011. Antimicro-
bial and antioxidant activities of endophytes from Tabebuia argentea and identifica-
tion of anticancer agent (lapachol). J. Med. Plants Res. 5, 3643–3652.
Ola, A.R.B., Thomy, D., Lai, D., Brӧtz-Oesterhelt, H., Proksch, P., 2013. Inducing secondary
metabolite production by the endophytic fungus Fusarium tricinctum through cocul-
ture with Bacillus subtilis. J. Nat. Prod. 76, 2094–2099.
Panaccione, D.G., Beaulieu, W.T., Cook, D., 2014. Bioactive alkaloids in vertically transmit-
ted fungal endophytes. Funct. Ecol. 28, 299–314.
Paranagama, P.A., Wijeratne, E.M.K., Gunatilaka, A.A.L., 2007. Uncovering biosynthetic
potential of plant-associated fungi: effect of culture conditions on metabolite
production by Paraphaeosphaeria quadriseptata and Chaetomium chiversii. J. Nat.
Prod. 70, 1939–1945.
Parthasarathy, R., Sathiyabama, M., 2014. Gymnemagenin-producing endophytic fungus
isolated from a medicinal plant Gymnema sylvestre R.Br. Appl. Biochem. Biotechnol.
172, 3141–3152.
Patwardhan, D.R.A., 2006. Research and Action in Natural Wealth Administration
(RANWA).
Pettit, R.K., 2010. Small‐molecule elicitation of microbial secondary metabolites. Microb.
Biotechnol. 4, 471–478.
Pu, X., Qu, X., Chen, F., Bao, J., Zhang, G., Luo, Y., 2013. Camptothecin-producing endophyt-
ic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation
conditions optimization for camptothecin production. Appl. Microbiol. Biotechnol.
97, 9365–9375.
Radman, R., Saez, T., Bucke, C., Keshavarz, T., 2003. Elicitation of plants and microbial cell
systems. Biotechnol. Appl. Biochem. 37, 91–102.
Ramachandra Rao, S., Ravishankar, G.A., 2002. Plant cell cultures: chemical factories of
secondary metabolites. Biotechnol. Adv. 20, 101–153.
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
Rodriguez, R.J., White Jr., J.F., Arnold, A.E., Redman, R.S., 2009. Fungal endophytes:
diversity and functional roles. New Phytol. 182, 314–330.
Sachin, N., Manjunatha, B.L., Mohana Kumara, P., Ravikanth, G., Shweta, S.,
Suryanarayanan, T.S., et al., 2013. Do endophytic fungi possess pathway genes for
plant secondary metabolites? Curr. Sci. 104, 178.
Scharf, D.H., Brakhage, A.A., 2013. Engineering fungal secondary metabolism: a roadmap
to novel compounds. J. Biotechnol. 163, 179–183.
Scherlach, K., Hertweck, C., 2009. Triggering cryptic natural product biosynthesis in
microorganisms. Org. Biomol. Chem. 7, 1753–1760.
Schügerl, K., Hubbuch, J., 2005. Integrated bioprocesses. Curr. Opin. Microbiol. 8, 294–300.
Schulz, B., Römmert, A.K., Dammann, U., Aust, H. Jür, Strack, D., 1999. The endophyte–host
interaction: a balanced antagonism? Mycol. Res. 103, 1275–1283.
Selosse, M.A., Le Tacon, F., 1998. The land flora: a phototroph–fungus partnership? Trends
Ecol. Evol. 13, 15–25.
Singh, M.P., Leighton, M.M., Barbieri, L.R., Roll, D.M., Urbance, S.E., Hoshan, L., et al., 2010.
Fermentative production of self-toxic fungal secondary metabolites. J. Ind. Microbiol.
Biotechnol. 37, 335–340.
Sirikantaramas, S., Yamazaki, M., Saito, K., 2009. A survival strategy: the coevolution of the
camptothecin biosynthetic pathway and self-resistance mechanism. Phytochemistry
70, 1894–1898.
Sohani, M.M., Schenk, P.M., Schultz, C.J., Schmidt, O., 2009. Phylogenetic and transcrip-
tional analysis of a strictosidine synthase‐like gene family in Arabidopsis thaliana
reveals involvement in plant defence responses. Plant Biol. 11, 105–117.
Soliman, S.S.M., Raizada, M.N., 2013. Interactions between co-habitating fungi elicit
synthesis of Taxol from an endophytic fungus in host Taxus plants. Front. Microbiol.
4, 1–14.
Soliman, S.S.M., Trobacher, C.P., Tsao, R., Greenwood, J.S., Raizada, M.N., 2013. A fungal
endophyte induces transcription of genes encoding a redundant fungicide pathway
in its host plant. BMC Plant Biol. 13, 93.
Spiering, M.J., Moon, C.D., Wilkinson, H.H., Schardl, C.L., 2005. Gene clusters for insecticidal
loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum. Genetics
169, 1403–1414.
Srivastava, S., Srivastava, A.K., 2007. Hairy root culture for mass-production of high value
secondary metabolites. Crit. Rev. Biotechnol. 27, 29–43.
Staniek, A., Woerdenbag, H.J., Kayser, O., 2009. Taxomyces andreanae: a presumed
paclitaxel producer demystified? Planta Med. 75, 1561–1566.
Stierle, A., Strobel, G., Stierle, D., 1993. Taxol and taxane production by Taxomyces
andreanae, an endophytic fungus of Pacific yew. Science 260, 214–216.
Stone, J.K., Bacon, C.W., White, J.F., 2000. An overview of endophytic microbes:
endophytism defined. In: Bacon, C.W., White, J.F. (Eds.), Microbial Endophytes.
Marcel Dekker, New York, pp. 3–30.
Strobel, G., Daisy, B., 2003. Bioprospecting for microbial endophytes and their natural
products. Microbiol. Mol. Biol. Rev. 67, 491–502.
Strobel, G.A., Ford, E., Li, J.Y., Sears, J., Sidhu, R.S., Hess, W.M., 1999. Seimatoantlerium
tepuiense gen. nov. A unique epiphytic fungus producing taxol from the Venezuelan
Guayana. System. Appl. Microbiol. 22, 426–433.
Strobel, G.A., Hess, W.M., Baird, G., Ford, E., Li, J.Y., Sidhu, R.S., 2001. Stegolerium kukenani
gen. nov. An endopyhtic, taxol producing fungus from the Roraima and Kukenan
Tepuis of Venezuela. Mycotaxon 78, 353–361.
Sun, Y., Luo, H., Li, Y., Sun, C., Song, J., Niu, Y., et al., 2011. Pyrosequencing of the
Camptotheca acuminata transcriptome reveals putative genes involved in
camptothecin biosynthesis and transport. BMC Genomics 12, 533.
Sun, J., Awakawa, T., Noguchi, H., Abe, I., 2012. Induced production of mycotoxins in an
endophytic fungus from the medicinal plant Datura stramonium L. Bioorg. Med.
Chem. Lett. 22, 6397–6400.
Taylor, L., 2000. Plant Based Drugs and Medicines. Raintree Nutr Inc., Carson City NV.
Taylor, L., 2005. The Healing Power of Rainforest Herbs: A Guide to Understanding and
Using Herbal Medicinals. Square One Publishers, New York.
Thammajaruk, N., Sriubolmas, N., Israngkul, D., Meevootisom, V., Wiyakrutta, S., 2011.
Optimization of culture conditions for mycoepoxydiene production by Phomopsis
sp. Hant25. J. Ind. Microbiol. Biotechnol. 38, 679–685.
Thiry, M., Cingolani, D., 2002. Optimizing scale-up fermentation processes. Trends
Biotechnol. 20, 103–105.
Tooker, J.F., Hanks, L.M., 2004. Trophic position of the endophytic beetle, Mordellistena
aethiops Smith (Coleoptera: Mordellidae). Environ. Entomol. 33, 291–296.
Trémouillaux-Guiller, J., Rohr, T., Rohr, R., Huss, V.A.R., 2002. Discovery of an endophytic
alga in Ginkgo biloba. Am. J. Bot. 89, 727–733.
Tudzynski, B., 2005. Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and
impact on biotechnology. Appl. Microbiol. Biotechnol. 66, 597–611.
Vasconsuelo, A., Boland, R., 2007. Molecular aspects of the early stages of elicitation of
secondary metabolites in plants. Plant Sci. 172, 861–875.
Venugopalan, A., Srivastava, S., 2015. Enhanced camptothecin production by ethanol
addition in the suspension culture of the endophyte, Fusarium solani. Bioresour.
Technol. 188, 251–257.
Verma, V.C., Lobkovsky, E., Gange, A.C., Singh, S.K., Prakash, S., 2011. Piperine production
by endophytic fungus Periconia sp. isolated from Piper longum L. J Antibiot (Tokyo)
64, 427–431.
Wang, Y., Guo, B., Miao, Z., Tang, K., 2007. Transformation of taxol‐producing endophytic
fungi by restriction enzyme‐mediated integration (REMI). FEMS Microbiol. Lett. 273,
253–259.
Wang, J., Lin, W., Wray, V., Lai, D., Proksch, P., 2013. Induced production of depsipeptides
by co-culturing Fusarium tricinctum and Fusarium begoniae. Tetrahedron Lett. 54,
2492–2496.
Wang, W.J., Li, D.Y., Li, Y.C., Hua, H.M., Ma, E.L., Li, Z.L., 2014a. Caryophyllene sesquiter-
penes from the marine-derived fungus Ascotricha sp. ZJ-M-5 by the One Strain-
Many Compounds Strategy. J. Nat. Prod. 77, 1367–1371.
 A. Venugopalan, S. Srivastava / Biotechnology Advances xxx (2015) xxx–xxx
Wang, X.J., Min, C.L., Ge, M., Zuo, R.H., 2014b. An endophytic sanguinarine-producing
fungus from Macleaya cordata, Fusarium proliferatum BLH51. Curr. Microbiol. 68,
336–341.
Weathers, P.J., Towler, M.J., Xu, J., 2010. Bench to batch: advances in plant cell culture for
producing useful products. Appl. Microbiol. Biotechnol. 85, 1339–1351.
Wei, Y., Zhou, X., Liu, L., Lu, J., Wang, Z., Yu, G., et al., 2010. An efficient transformation
system of taxol-producing endophytic fungus EFY-21 (Ozonium sp.). Afr. J. Biotechnol.
9, 1726–1733.
Wilson, S.A., Roberts, S.C., 2012. Recent advances towards development and commercial-
ization of plant cell culture processes for the synthesis of biomolecules. Plant
Biotechnol. J. 10, 249–268.
Xiong, Z.Q., Yang, Y.Y., Zhao, N., Wang, Y., 2013. Diversity of endophytic fungi and screen-
ing of fungal paclitaxel producer from Anglojap yew, Taxus × media. BMC Microbiol.
13, 71.
Xu, F., Tao, W., Cheng, L., Guo, L., 2006a. Strain improvement and optimization of the
media of taxol-producing fungus Fusarium maire. Biochem. Eng. J. 31, 67–73.
Xu, F., Tao, W.Y., Cheng, L., Guo, L.J., 2006b. Formation and regeneration of protoplasts of
taxol-producing endophytic fungus Fusarium maire. J. Food Sci. Biotechnol. 25, 20–24.
Xu, L.J., Liu, Y.S., Zhou, L.G., Wu, J.Y., 2009. Enhanced beauvericin production with in situ
adsorption in mycelial liquid culture of Fusarium redolens Dzf2. Process Biochem.
44, 1063–1067.
Xu, L.J., Liu, Y.S., Zhou, L.G., Wu, J.Y., 2010. Optimization of a liquid medium for beauvericin
production in Fusarium redolens Dzf2 mycelial culture. Biotechnol. Bioprocess Eng. 15,
460–466.
Yamazaki, Y., Kitajima, M., Arita, M., Takayama, H., Sudo, H., Yamazaki, M., et al., 2004. Bio-
synthesis of camptothecin. In silico and in vivo tracer study from [1–13C] glucose.
Plant Physiol. 134, 161–170.
Yamin, W., Lu, L., Xuanwei, Z., Juan, L., Xiaofen, S., Kexuan, T., 2012. Engineering taxol
biosynthetic pathway for improving taxol yield in taxol-producing endophytic fungus
EFY-21 (Ozonium sp.). Afr. J. Biotechnol. 11, 9094–9101.
Yang, X.L., Awakawa, T., Wakimoto, T., Abe, I., 2013. Induced production of novel
prenyldepside and coumarins in endophytic fungi Pestalotiopsis acaciae. Tetrahedron
Lett. 54, 5814–5817.
Yang, H., Wang, Y., Zhang, Z., Yan, R., Zhu, D., 2014a. Whole-genome shotgun assembly
and analysis of the genome of Shiraia sp. strain Slf14, a novel endophytic fungus
producing huperzine A and hypocrellin A. Genome Announc. 2, e00011–e00014.
Yang, X.L., Huang, L., Ruan, X.L., 2014b. Epigenetic modifiers alter the secondary metabo-
lite composition of a plant endophytic fungus, Pestalotiopsis crassiuscula obtained
from the leaves of Fragaria chiloensis. J. Asian Nat. Prod. Res. 16, 412–417.
Yang, Y., Zhao, H., Barrero, R.A., Zhang, B., Sun, G., Wilson, I.W., et al., 2014c. Genome se-
quencing and analysis of the paclitaxel-producing endophytic fungus Penicillium
aurantiogriseum NRRL 62431. BMC Genomics 15, 69.
Yin, H., Chen, J.L., 2008. Sipeimine-producing endophytic fungus isolated from Fritillaria
ussuriensis. Z. Naturforsch. C 63, 789.
Yin, H., Chen, J., 2011. The fermentation conditions of a sipeimine producing endophytic
fungus isolated from Fritillaria ussuriensis. J. Northwest Univ. (Nat. Sci. Ed.) 2, 18.
Yin, H., Sun, Y.H., 2011. Vincamine-producing endophytic fungus isolated from Vinca
minor. Phytomedicine 18, 802–805.
Yin, H., Zhao, Q., Sun, F.M., An, T., 2009. Gentiopicrin-producing endophytic fungus
isolated from Gentiana macrophylla. Phytomedicine 16, 793–797.
You, X., Feng, S., Luo, S., Cong, D., Yu, Z., Yang, Z., et al., 2013. Studies on a rhein-producing
endophytic fungus isolated from Rheum palmatum L. Fitoterapia 85, 161–168.
Yu, T.W., Bai, L., Clade, D., Hoffmann, D., Toelzer, S., Trinh, K.Q., et al., 2002. The biosynthetic
gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema
pretiosum. Proc. Natl. Acad. Sci. U. S. A. 99, 7968–7973.
Zhang, P., Zhou, P., Yu, L., 2009a. An endophytic taxol‐producing fungus from Taxus ×
media, Aspergillus candidus MD3. FEMS Microbiol. Lett. 293, 155–159.
Zhang, P., Zhou, P.P., Yu, L.J., 2009b. An endophytic taxol-producing fungus from Taxus
media, Cladosporium cladosporioides MD2. Curr. Microbiol. 59, 227–232.
Zhang, P., Liu, T.T., Zhou, P.P., Li, S.T., Yu, L.J., 2011a. Agrobacterium tumefaciens-mediated
transformation of a taxol-producing endophytic fungus, Cladosporium cladosporioides
MD2. Curr. Microbiol. 62, 1315–1320.
Please cite this article as: Venugopalan, A., Srivastava, S., Endophytes as in vitro production platforms of high value plant secondary metabolites,
Biotechnol Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.07.004
15
Zhang, W., Wang, M., Huang, Y., Chea, S.K.P., Zheng, Z., Qian, X., et al., 2011b. New and
highly efficient methodology for screening high-yield strains of cytotoxic
deacetylmycoepoxydiene (DAM). Lett. Appl. Microbiol. 52, 441–447.
Zhao, K., Zhou, D.P., Ping, W.X., Ge, J., 2004. Study on the preparation and regeneration of
protoplast from taxol-producing fungus Nodulisporium sylviforme. Nat. Sci. 2, 52–59.
Zhao, J., Davis, L.C., Verpoorte, R., 2005a. Elicitor signal transduction leading to production
of plant secondary metabolites. Biotechnol. Adv. 23, 283–333.
Zhao, K., Ping, W., Ma, X., Liu, J., Zhou, D., 2005b. Breeding of high-yield strain of taxol by
mutagenesis of protoplast and primary discussion of genetic differences between
mutants and their parent strain. Wei Sheng Wu Xue Bao 45, 355–358.
Zhao, J., Zhou, L., Wang, J., Shan, T., Zhong, L., Liu, X., et al., 2010. Endophytic fungi for pro-
ducing bioactive compounds originally from their host plants. Curr. Res. Technol.
Educ. Trop. Appl. Microbiol. Microb. Biotechnol. 1, 567–576.
Zhao, J., Li, Y., Shan, T., Mou, Y., Zhou, L., 2011a. Enhancement of diepoxin ζ production
with in situ resin adsorption in mycelial liquid culture of the endophytic fungus
Berkleasmium sp. Dzf12 from Dioscorea zingiberensis. World J. Microbiol. Biotechnol.
27, 2753–2758.
Zhao, J., Shan, T., Mou, Y., Zhou, L., 2011b. Plant-derived bioactive compounds produced
by endophytic fungi. Mini Rev. Med. Chem. 11, 159–168.
Zhao, J., Zheng, B., Li, Y., Shan, T., Mou, Y., Lu, S., et al., 2011c. Enhancement of diepoxin ζ
production by yeast extract and its fractions in liquid culture of Berkleasmium-like
endophytic fungus Dzf12 from Dioscorea zingiberensis. Molecules 16, 847–856.
Zhao, K., Li, Z., Ge, N., Li, X., Wang, X., Zhou, D., 2011d. Investigation of fermentation con-
ditions and optimization of medium for taxol production from taxol-producing fungi.
J. Med. Plants Res. 5, 6528–6535.
Zhao, K., Sun, L., Wang, X., Li, X., Zhou, D., 2011e. Screening of high taxol producing fungi
by mutagenesis and construction of subtracted cDNA library by suppression
subtracted hybridization for differentially expressed genes. Wei Sheng Wu Xue Bao
51, 923–933.
Zhao, J., Wang, X., Sun, W., Mou, Y., Peng, Y., Zhou, L., 2013a. Medium optimization for
palmarumycin C13 production in liquid culture of endophytic fungus Berkleasmium
sp. Dzf12 using response surface methodology. Electron. J. Biotechnol. 16, 16.
Zhao, K., Sun, L., Ma, X., Li, X., Wang, X., Ping, W., et al., 2013b. Improved taxol production
in Nodulisporium sylviforme derived from inactivated protoplast fusion. Afr.
J. Biotechnol. 10, 4175–4182.
Zhao, X.M., Wang, Z.Q., Shu, S.H., Wang, W.J., Xu, H.J., Ahn, Y.J., et al., 2013c. Ethanol and
methanol can improve huperzine A production from endophytic Colletotrichum
gloeosporioides ES026. PLoS ONE 8, e61777.
Zhou, D.P., Sun, J.Q., Yu, H.Y., Ping, W.X., Zheng, X.Q., 2001. Nodulisporium, a genus new to
China. Mycosystema 20, 277–278.
Zhou, D., Zhao, K., Ping, W., Ge, J., Ma, X., Jun, L., 2005. Study on the mutagenesis of
protoplasts from taxol-producing fungus Nodulisporium sylviforme. J. Am. Sci. 1, 62.
Zhou, X., Wang, Z., Jiang, K., Wei, Y., Lin, J., Sun, X., et al., 2007. Screening of taxol-
producing endophytic fungi from Taxus chinensis var. mairei. Appl. Biochem.
Microbiol. 43, 439–443.
Zhou, X., Wei, Y., Zhu, H., Wang, Z., Lin, J., Liu, L., et al., 2008. Protoplast formation,
regeneration and transformation from the taxol-producing fungus Ozonium sp. Afr.
J. Biotechnol. 7, 2017–2024.
Zhou, X., Zhu, H., Liu, L., Lin, J., Tang, K., 2010. A review: recent advances and future
prospects of taxol-producing endophytic fungi. Appl. Microbiol. Biotechnol. 86,
1707–1717.
Zhu, F., Lin, Y., 2006. Marinamide, a novel alkaloid and its methyl ester produced by the
application of mixed fermentation technique to two mangrove endophytic fungi
from the South China Sea. Chin. Sci. Bull. 51, 1426–1430.
Zhu, F., Lin, Y., Ding, J., Wang, X., Huang, L., 2007. Secondary metabolites of two marine-
derived mangrove endophytic fungi (strain nos. 1924# and 3893#) by mixed
fermentation. Chem. Ind. For. Prod. 27, 8–10.
Zu, Y.G., Tang, Z.H., Yu, J.H., Liu, S.G., Wang, W., Guo, X.R., 2003. Different responses of
camptothecin and 10-hydroxycamptothecin to heat shock in Camptotheca acuminata
seedlings. Acta Bot. Sin. 45, 809–814.