Modulation of genetic clusters for synthesis of bioactive molecules in fungal endophytes: A review

Abstract

Novel drugs with unique and targeted mode of action are very much need of the hour to treat and manage severe multidrug infections and other life-threatening complications. Though natural molecules have proved to be effective and environmentally safe, the relative paucity of discovery of new drugs has forced us to lean towards synthetic chemistry for developing novel drug molecules. Plants and microbes are the major resources that we rely upon in our pursuit towards discovery of novel compounds of pharmacological importance with less toxicity. Endophytes, an eclectic group of microbes having the potential to chemically bridge the gap between plants and microbes, have attracted the most attention due to their relatively high metabolic versatility. Since continuous large scale supply of major metabolites from microfungi and especially endophytes is severely impeded by the phenomenon of attenuation in axenic cultures, the major challenge is to understand the regulatory mechanisms in operation that drive the expression of metabolic gene clusters of pharmaceutical importance. This review is focused on the major regulatory elements that operate in filamentous fungi and various combinatorial multi-disciplinary approaches involving bioinformatics, molecular biology, and metabolomics that could aid in large scale synthesis of important lead molecules.

Full text

Microbiological
Research
182
(2016)
125–140
Contents
lists
available
at
ScienceDirect
Microbiological
Research
j
ourna
l
h
om
epage:
www.elsevier.com/locate/micres
Modulation
of
genetic
clusters
for
synthesis
of
bioactive
molecules
in
fungal
endophytes:
A
review
V.B.
Deepika,
T.S.
Murali∗,
K.
Satyamoorthy
Division
of
Biotechnology,
School
of
Life
Sciences,
Manipal
University,
Manipal
576104,
India
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
5
August
2015
Received
in
revised
form
21
October
2015
Accepted
26
October
2015
Available
online
29
October
2015
Keywords:
Endophytes
Secondary
metabolites
Orphan
gene
clusters
Gene
activation
Attenuation
Regulation
a
b
s
t
r
a
c
t
Novel
drugs
with
unique
and
targeted
mode
of
action
are
very
much
need
of
the
hour
to
treat
and
manage
severe
multidrug
infections
and
other
life-threatening
complications.
Though
natural
molecules
have
proved
to
be
effective
and
environmentally
safe,
the
relative
paucity
of
discovery
of
new
drugs
has
forced
us
to
lean
towards
synthetic
chemistry
for
developing
novel
drug
molecules.
Plants
and
microbes
are
the
major
resources
that
we
rely
upon
in
our
pursuit
towards
discovery
of
novel
compounds
of
pharmacological
importance
with
less
toxicity.
Endophytes,
an
eclectic
group
of
microbes
having
the
potential
to
chemically
bridge
the
gap
between
plants
and
microbes,
have
attracted
the
most
attention
due
to
their
relatively
high
metabolic
versatility.
Since
continuous
large
scale
supply
of
major
metabolites
from
microfungi
and
especially
endophytes
is
severely
impeded
by
the
phenomenon
of
attenuation
in
axenic
cultures,
the
major
challenge
is
to
understand
the
regulatory
mechanisms
in
operation
that
drive
the
expression
of
metabolic
gene
clusters
of
pharmaceutical
importance.
This
review
is
focused
on
the
major
regulatory
elements
that
operate
in
filamentous
fungi
and
various
combinatorial
multi-disciplinary
approaches
involving
bioinformatics,
molecular
biology,
and
metabolomics
that
could
aid
in
large
scale
synthesis
of
important
lead
molecules.
©
2015
Elsevier
GmbH.
All
rights
reserved.
Contents
1.
Introduction
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2.
Interaction
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host
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3.
Gene
clustering—an
important
aspect
for
functionally
important
genes
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3.1.
Significance
of
clustering
of
genes
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127
3.2.
Gene
clusters
in
fungi
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127
4.
Genetic
make-up
of
plant
secondary
metabolism
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127
5.
Genetic
make-up
of
fungal
secondary
metabolism
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128
6.
Identifying
fungal
gene
clusters
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128
7.
Regulation
of
secondary
metabolism
in
fungi
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128
7.1.
G-protein
signaling
in
filamentous
fungi
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7.2.
Fungal
development
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secondary
metabolism
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129
7.3.
Velvet
complex
as
a
regulator
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129
7.4.
Role
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7.5.
Regulation
through
environmental
stimuli
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130
7.6.
Pathway
specific
regulation
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130
7.6.1.
In-cluster
genes
encoding
pathway-specific
regulators
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130
7.6.2.
Position
of
the
regulatory
gene
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131
7.6.3.
Cross-talk
between
gene
clusters
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131
7.7.
Global
regulation
in
fungi
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131
∗Corresponding
author.
Fax:
+91
820
2571919.
E-mail
address:
murali.ts@manipal.edu
(T.S.
Murali).
http://dx.doi.org/10.1016/j.micres.2015.10.009
0944-5013/©
2015
Elsevier
GmbH.
All
rights
reserved.

126
V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
7.8.
Chromatin
structure
and
histone
modifications
in
regulation
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132
7.8.1.
Chromatin-mediated
regulation
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132
7.8.2.
Histone
methylation
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132
7.8.3.
Histone
acetylation—an
essential
mark
for
SM
gene
activation.
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.133
8.
Modifications
to
overcome
metabolic
toxicity
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133
9.
Microfungi
as
alternatives
for
plant
bioactive
compound
synthesis
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134
10.
Activation
of
orphan
biosynthetic
gene
clusters
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134
11.
Current
approaches
in
activation
of
gene
clusters.
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.135
11.1.
Gene
deletions
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135
11.2.
Modulation
in
epigenetic
mechanisms
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135
11.3.
Proteomic
approach
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135
11.4.
Genome
mining.
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.135
12.
Heterologous
hosts
for
cloning
and
expression
of
fungal
metabolites
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135
13.
Conclusion
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136
Acknowledgements
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136
References
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136
1.
Introduction
Nature
remains
a
munificent
source
of
important
natural
prod-
ucts
and
has
helped
mankind
to
overcome
several
life-threatening
complications.
A
vast
majority
of
chemical
diversity
in
nature
is
derived
from
plants
and
microbes
that
remain
a
valuable
resource
for
novel
lead
discovery
by
pharmaceutical
industries.
Plants
with
ethno-pharmaceutical
importance
have
been
vigorously
screened
for
medicinally
important
lead
molecules.
Though
pharmaceutical
industries
have
recently
shifted
their
focus
on
synthetic
molecules,
many
‘safe’
and
long-lasting
drugs
are
derived
from
natural
sources.
The
underlying
structural
complexity
of
natural
drugs
makes
the
chemical
synthesis
of
these
compounds
difficult
and
hence
plants
are
considered
a
promising
source
in
obtaining
compounds
with
high
biological
efficiency.
Isolation,
purification
and
characteriza-
tion
of
structurally
complex
lead
molecules
in
adequate
yield
from
plant
sources
remain
a
major
bottleneck
(Wu
and
Chappell,
2008).
Microbes
have
recently
become
the
primary
targets
in
screen-
ing
programs
and
yet
plant-associated
microbes
have
remained
largely
unexplored
for
their
chemical
diversity.
Fungal
endophytes
exist
in
close
symbiotic
association
with
plants
(Rodriguez
et
al.,
2009)
and
have
gained
much
attention
recently
due
to
their
diver-
sity
and
interesting
biotechnological
potential
(Hyde
and
Soytong,
2008).
They
are
endowed
with
a
repertoire
of
enzymes
that
aid
in
biosynthesis
of
structurally
diverse
and
complex
molecules
that
are
often
difficult
to
mimic
(Kusari
and
Spiteller,
2011).
The
sci-
entific
community
firmly
shifted
its
attention
towards
endophytic
microbes
when
it
was
discovered
that
the
endophytic
fungus
Tax-
omyces
andreanae
from
yew
plants
could
produce
paclitaxel
(Stierle
et
al.,
1993)
and
several
other
studies
also
showed
that
endophytes
have
the
ability
to
produce
host-derived
metabolites
of
importance
(Table
1).
Bioprospecting
of
endophytes
offers
significant
promise
in
the
discovery
of
plant-associated
secondary
metabolites
(SMs)
and
their
analogs
or
novel
lead
compounds
for
synthetic
modifica-
tions
towards
therapeutic
applications.
The
major
hindrance
in
the
large
scale
biosynthesis
of
SMs
usually
encoded
by
gene
modules
or
operons
is
that
regulation
mechanisms
involved
at
the
molec-
ular
level
still
needs
to
be
understood
fully.
We
focus
our
review
on
the
principal
underlying
molecular
mechanisms
involved
in
the
initiation
and
regulation
of
plant
SM
biosynthesis
in
fungi
and
in
particular
fungal
endophytes.
2.
Interaction
between
host
and
endophyte
The
critical
aspect
in
understanding
the
regulation
mechanism
is
to
recognize
the
interaction
between
the
possible
symbiotic
partners
–
the
host
and
the
endophyte
–
as
plants
are
known
to
be
systemically
colonized
by
diverse
endophytes
albeit
with-
out
any
external
symptoms.
Host
plants
from
different
continents
and
geographical
origin
are
known
to
harbor
diverse
endophytes
(Arnold
and
Lutzoni,
2007;
Carroll,
1995;
Hoffman
and
Arnold,
2008).
Metagenome
analysis
from
the
inner
tissues
of
healthy
plants
indicates
the
presence
of
diverse
group
of
microbial
organ-
isms
living
within
the
plant
host
(Jumpponen
and
Jones,
2009).
Host
genetic
factors
may
critically
influence
the
structure
and
function
of
plant
associated
microbiomes
and
especially
play
an
influencing
role
in
selecting
vertically
transmitted
endophytes
(Ahlholm
et
al.,
2002;
Hardoim
et
al.,
2011).
Endophytes
seem
to
have
successfully
adapted
themselves
to
overcome
host
immune
system
to
form
pop-
ulations
in
the
internal
tissues
of
the
host.
Communication
within
microbial
endophytic
communities
and
with
the
host
would
have
an
impact
on
physiological
processes
of
the
plant
including
plant
metabolite
synthesis.
Endophytic
microbiomes
are
known
to
significantly
influence
host
performance
especially
under
stressed
conditions
(Hamilton
and
Bauerle,
2012).
Fungal
endophytes
may
mediate
functioning
of
plant
micro-ecosystem
by
critically
altering
the
responses
of
the
plant
with
respect
to
environmental
changes
(Friesen
et
al.,
2011).
It
is
not
unusual
to
observe
that
both
the
plant
and
endo-
phytes
synthesize
an
extensive
collection
of
similar
metabolites
having
common
precursors.
Common
isoprenoids
precursors,
hor-
mones,
quinones
and
other
SMs
with
major
role
in
plant
protection
and
communication
are
also
widely
synthesized
in
archaea
and
eubacteria
(Kirby
and
Keasling,
2009).
Howitz
and
Sinclair
(2008)
hypothesized
that
microorganisms
might
sense
any
stress-induced
molecules
from
plants
and
homologous
gene
clusters
present
in
plants
and
microorganisms
may
get
cross-activated
when
plants
come
under
some
stressful
conditions
as
in
a
pathogen
attack.
One
of
the
recognized
case
in
point
is
the
synthesis
of
Taxol
from
yew
plant
and
from
several
endophytic
fungi
isolated
from
this
plant
(Zhou
et
al.,
2010a).
3.
Gene
clustering—an
important
aspect
for
functionally
important
genes
A
universal
trait
of
bacterial
genomes
is
the
organization
of
genes
into
functional
groups
as
operons
consisting
of
clusters
of
genes
with
related
functions
that
are
transcribed
as
a
block
(Jacob
and
Monod,
1961;
Koonin,
2009;
Rocha,
2008).
Random
arrangement
of
genes
was
considered
the
norm
in
eukaryotes
with
active
operons
rarely
being
reported
with
a
few
notable
excep-
tions
(Blumenthal
and
Gleason,
2003).
Recent
genetic
studies
in
eukaryotes
suggest
presence
of
non-homologous
gene
clusters
that
are
functionally
associated
(Hurst
et
al.,
2004;
Osbourn
and
Field,
2009).
These
functionally
associated
genes
without
any
sequence

V.B.
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/
Microbiological
Research
182
(2016)
125–140
127
Table
1
Endophytic
filamentous
fungi
reported
to
produce
host-derived
metabolites.
Metabolite
Endophytic
fungus
Host
plant
References
Taxol
Taxomyces
andreanae
Taxus
brevifolia
Stierle
et
al.
(1993)
Vinblastine
Alternaria
sp.
Catharanthus
roseus
Guo
et
al.
(1998)
Vincristine
Fusarium
oxysporum
Catharanthus
roseus
Zhang
et
al.
(2000)
Diosgenin
Cephalosporium
sp.
Paris
polyphylla
var.
yunnanensis
Zhou
et
al.
(2004)
Camptothecin
Entrophospora
infrequens
Nothapodytes
foetida
Puri
et
al.
(2005)
Huperzine
A
Acremonium
sp.
Huperzia
serrata
Li
et
al.
(2007)
Podophyllotoxin
Fusarium
oxysporum
Juniperus
recurva
Kour
et
al.
(2008)
Hypericin
Chaetomium
globosum
Hypericum
perforatum
Kusari
et
al.
(2008)
Camptothecin
Fusarium
solani
Apodytes
dimidiata
Shweta
et
al.
(2010)
Rohitukine
Fusarium
proliferatum
Dysoxylum
binectariferum
Kumara
et
al.
(2012)
Azadirachtin
Eupenicillium
parvum
Azadirachta
indica
Kusari
et
al.
(2012a)
Podophyllotoxin
Alternaria
sp.
Sinopodophyllum
emodi
Liang
et
al.
(2015)
similarity
are
widespread
all
over
the
genome
and
are
co-regulated
but
not
transcribed
as
a
single
unit
as
in
prokaryotes.
3.1.
Significance
of
clustering
of
genes
Gene
clustering
aids
in
synchronized
transcriptional
regula-
tion
by
sharing
long-distance
regulatory
elements
or
by
regulating
changes
in
chromatin
organization
(Hurst
et
al.,
2004;
Osbourn,
2010a,b;
Sproul
et
al.,
2005).
It
has
been
evident
in
yeasts,
filamen-
tous
fungi
and
mammals
that
expression
of
gene
cluster
is
linked
to
histone
modifications
brought
about
by
chromatin
remodeling
complexes
(Osbourn
and
Field,
2009).
The
primary
advantage
of
gene
clustering
is
co-inheritance
and
co-regulation
of
constructive
combinations
of
alleles
at
functionally
important
loci.
The
physical
proximity
of
entire
cluster
might
be
obligatory
for
the
production
of
end
product
in
a
pathway
and
thus
favor
an
assortment
of
genomic
rearrangements
that
keep
pathway
genes
together
(Nei,
2003).
Dis-
ruption
of
genes
required
for
SM
synthesis
may
not
only
lead
to
loss
of
the
bioactive
end
product
with
a
major
protective
role
but
also
cause
accumulation
of
toxic
pathway
intermediates.
In
addi-
tion
to
harmful
effects
caused
by
this
pathway
disruption,
it
can
also
have
compromised
effect
towards
pest
and
disease
resistance
and
hence
improve
cluster
selection.
Varied
levels
of
transcriptional
co-
regulation
might
be
achieved
in
clustered
genes
(Hurst
et
al.,
2004;
Sproul
et
al.,
2005).
A
rather
extreme
approach
towards
this
trend
is
achieved
by
fusion
of
these
genes
to
obtain
a
single
gene
product
(Gross
et
al.,
2006;
Hawkins,
1987;
Zhang
and
Smith,
1998).
3.2.
Gene
clusters
in
fungi
Lawrence
and
Roth
(1996)
proposed
the
‘selfish
operons’
theory
to
explain
gene
clustering
in
bacteria.
According
to
them,
horizontal
gene
transfer
(HGT)
promotes
clustering
of
genes
as
rearrange-
ment
of
genetic
material
might
bring
genes
closer
that
will
enhance
co-mobilization
of
genes.
Conversely,
it
can
also
be
argued
that
the
arrangement
of
genes
in
clusters
provide
enough
evidence
for
HGT
(Rosewich
and
Kistler,
2000).
In
filamentous
fungi
such
as
Aspergillus
nidulans,
Penicillium
chrysogenum,
Penicillium
nal-
giovense
and
Penicillium
notatum,
the
clustering
of
genes
coding
for
␤-lactam
antibiotic
penicillin
synthesis
are
similar
to
clus-
ters
found
in
Acremonium
chrysogenum
for
␤-lactam
cephalosporin
biosynthesis
(Gutierrez
et
al.,
1999).
Similar
gene
clusters
are
found
in
Streptomyces
spp.
and
gram-negative
eubacteria
for
penicillin
and
cephalosporin
biosynthesis
(Aharonowitz
et
al.,
1992).
In
both
the
antibiotics,
a
common
gene
pcbC
is
required
for
synthesis
of
isopenicillin-N-synthase
enzyme
coding
for
␤-lactam
ring
forma-
tion.
Very
high
similarity
in
both
amino
acid
and
DNA
sequences
has
been
observed
in
the
fungus
P.
chrysogenum,
gram-negative
bac-
terium
Flavobacterium
and
gram-positive
bacterium
Streptomyces
griseus
(Aharonowitz
et
al.,
1992).
Quite
a
few
naturally
occur-
ring
strains
and
mutants
of
P.
chrysogenum
were
found
to
harbor
the
pcbC
gene
but
differed
only
in
the
ability
to
synthesize
peni-
cillin.
It
was
identified
that
the
strain
which
produces
penicillin
at
normal
level
harbors
a
single
copy
of
the
cluster
gene,
whereas
over-producing
strains
showed
tandem
amplification
(Fierro
et
al.,
1995).
The
clusters
were
flanked
by
a
conserved
hexanucleotide
sequence
repeat
TTTACA
while
mutants
were
found
to
have
the
penicillin
biosynthetic
unit
deleted
(Fierro
et
al.,
1996).
Genes
arranged
in
clusters
mediating
pathogenicity
in
phy-
topathogenic
fungi
have
also
been
reported.
Fusarium
spp.
harbor
gene
clusters
involved
in
synthesis
of
gibberellins
(Tudzynski
and
Holter,
1998),
mycotoxins
and
trichothecenes
(Hohn
et
al.,
1993).
Species
of
Cochliobolus
carry
TOX
locus
which
codes
for
a
cyclic
tetrapeptide
HC-Toxin
required
for
its
pathogenicity
towards
maize.
This
locus
is
amplified
in
pathogenic
strains
and
is
com-
pletely
absent
in
strains
that
fail
to
produce
toxin
while
mutations
in
this
locus
led
to
non-pathogenicity
(Panaccione
et
al.,
1992).
In
Nectria
haematococca,
three
genes
part
of
PEP
cluster
located
in
supernumerary
chromosomes
are
implicated
in
causing
disease
on
pea
plants.
The
PEP
cluster
varies
from
other
portions
of
the
genome
in
several
features
including
unique
usage
of
codons,
GC
content
and
the
presence
of
transposons
(Han
et
al.,
2001).
Dis-
pensable
chromosomes
and
dispensable
portions
of
chromosomes
have
been
reported
from
several
fungal
genome
sequences
and
the
presence
of
additional
elements
of
genome
namely
B
chromo-
somes,
supernumerary
chromosomes,
dispensable
chromosomes
and
minichromosomes
have
been
attributed
to
add
to
its
complex-
ity.
4.
Genetic
make-up
of
plant
secondary
metabolism
Secondary
metabolites
are
low-molecular-weight
structurally
heterogeneous
molecules
with
no
direct
role
in
the
growth
of
the
host
that
produce
them.
The
genes
involved
in
secondary

128
V.B.
Deepika
et
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/
Microbiological
Research
182
(2016)
125–140
Table
2
List
of
some
of
the
metabolites
synthesized
from
endophytic
filamentous
fungi.
Metabolite
Endophytic
fungus
Host
References
Torreyanic
acid
Pestalotiopsis
microspora
Taxus
taxifolia
Lee
et
al.
(1996)
Cytosporones
Cytospora
sp. Conocarpus
erecta Clardy
et
al.
(2000)
Cytoskyrins
A
and
B
Cytospora
sp.
Conocarpus
erecta
Brady
et
al.
(2000)
Isopestacin
Pestalotiopsis
microspora
Terminalia
morobensis
Strobel
et
al.
(2002)
Pestacin
Pestalotiopsis
microspora
Terminalia
morobensis
Harper
et
al.
(2003)
Periconicins
Periconia
sp.
Taxus
cuspidata
Kim
et
al.
(2004)
Graphislactone
A
Cephalosporium
sp.
Trachelospermum
jasminoides
Song
et
al.
(2005)
Trichodermin
Trichoderma
harzianum
Ilex
cornuta
Chen
et
al.
(2007)
Phomoenamide
and
Phomonitroester Phomopsis
sp. Garcinia
dulcis Rukachaisirikul
et
al.
(2008)
Sordaricin
Xylaria
sp.
Garcinia
dulcis
Pongcharoen
et
al.
(2008)
Labdane
and
tetranorlabdane
diterpenoids
Botryosphaeria
sp.
Maytenus
hookeri
Yuan
et
al.
(2009)
Ergoflavin
Unidentified
fungus
Mimusops
elengi
Deshmukh
et
al.
(2009)
Asporyzin
C
Aspergillus
oryzae
Heterosiphonia
japonica
(marine
red
alga)
Qiao
et
al.
(2010)
Botryorhodines
Botryosphaeria
rhodina
Bidens
pilosa
Abdou
et
al.
(2010)
Phomenone
Xylaria
sp.
Piper
aduncum
Silva
et
al.
(2010)
Serobactin
A,B,C
Herbaspirillum
seropedicae
Grass
crops
Rosconi
et
al.
(2013)
Dalsymbiopyrone
Daldinia
hawksworthii
Xiphydria
prolongata
(woodwasp)
Pazoutova
et
al.
(2013)
metabolism
in
plants
are
usually
clustered.
DIMBOA
cluster
in
maize
was
the
first
plant
SM
pathway
cluster
to
be
discovered
(Frey
et
al.,
1997).
Other
major
gene
clusters
include
thalianol
cluster
that
synthesizes
triterpenes
in
Arabidopsis
thaliana,
ave-
nacin
cluster
in
Avena
spp.
(Mugford
et
al.,
2009;
Mylona
et
al.,
2008;
Papadopoulou
et
al.,
1999;
Qi
et
al.,
2004;
Qin
et
al.,
2010),
momilactone
and
phytocassane
clusters
in
Oryza
sativa
(Sakamoto
et
al.,
2004;
Swaminathan
et
al.,
2009)
and
cluster
for
synthesis
of
the
cyclic
hydroxamic
acids
2,4-dihydroxy-1,4-benzoxazin-3-
one
(DIBOA)
and
2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
(DIMBOA)
in
maize
(Frey
et
al.,
1997;
Jonczyk
et
al.,
2008;
von
Rad
et
al.,
2001).
These
clusters
play
a
major
role
in
the
synthesis
of
defense
compounds
in
the
host
which
confers
pest
and
dis-
ease
resistance
(Shimura
et
al.,
2007).
Though
the
significance
of
thalianol
cluster
is
yet
to
be
deciphered
completely,
it
is
widely
believed
to
have
a
vital
ecological
significance
due
to
its
increased
rate
of
conservation
in
different
A.
thaliana
accessions
(Field
and
Osbourn,
2008).
5.
Genetic
make-up
of
fungal
secondary
metabolism
In
microbes,
it
is
widely
believed
that
SMs
are
chemical
sig-
naling
entities
synthesized
for
communication
and
play
a
major
role
in
inhibition
of
competitors
(Brakhage
and
Schroeckh,
2011;
Yim
et
al.,
2007).
The
genes
coding
for
SM
biosynthesis
in
fungi
are
also
arranged
in
clusters
(Smith
et
al.,
1990)
that
can
span
more
than
10
kb
(Trail
et
al.,
1995),
although
there
are
a
few
exceptions
(Lo
et
al.,
2012).
The
arrangement
of
SM
genes
in
clusters
limits
the
use
of
fermentation
technique
as
a
viable
and
suitable
alterna-
tive
towards
large-scale
production
of
several
metabolites
(Wu
and
Chappell,
2008).
These
clusters
usually
code
for
enzyme
complexes
such
as
the
non-ribosomal
peptide
synthetases
[NRPS]
or
polyke-
tide
synthases
[PKS]
that
consist
of
several
domains
and
modules
with
defined
functions
(Brakhage
et
al.,
2009a;
Hertweck,
2009a;
Strieker
et
al.,
2010).
These
multimodular
enzymes
exhibit
high
similarity
in
their
architecture
and
mechanisms
involved
in
the
product
assembly
thus
aiding
in
recruitment
of
different
substrates
(Cane
and
Walsh,
1999;
Hertweck,
2009a).
To
synthesize
the
struc-
tural
backbone
of
the
respective
SMs,
PKS
and
NRPS
use
malonyl
groups
and
amino
acids
or
their
derivatives
as
the
building
blocks
(Brakhage
and
Schroeckh,
2011;
Hertweck,
2009a).
Polyketides
(PK)
or
non-ribosomal
peptides
(NRP)
form
a
back-
bone
for
most
of
the
secondary
metabolites.
Some
of
the
common
derivatives
of
NRP
include
clinically
important
antibiotics
such
as
penicillin
and
cephalosporin
and
immune
suppressants
like
cyclosporine,
while
lovastatin
is
derived
from
the
polyketide
scaffold
(Brakhage,
1998;
Hoffmeister
and
Keller,
2007).
Some
compounds
fall
under
the
mixed
PKS-NRP
hybrid
origin
like
aspyridones
(Bergmann
et
al.,
2007)
while
others
like
gibberellins
(terpene
derivatives)
and
oxylipins
(fatty
acid
derivatives)
result
from
alternative
pathways
(Berdy,
2005).
As
mentioned
earlier,
endophytes
have
received
much
attention
in
the
recent
past
due
to
their
high
metabolic
versatility
and
several
reviews
have
focused
their
attention
of
secondary
metabolites
produced
by
endophytes
(Aly
et
al.,
2010;
Guo
et
al.,
2008;
Schulz
et
al.,
2002;
Suryanarayanan
et
al.,
2009;
see
Tan
and
Zou,
2001).
While
fungal
endophytes
have
been
vigorously
screened
for
their
potential
to
elicit
host-derived
phytochemicals,
other
reports
clearly
indicate
that
endophytic
filamentous
fungi
also
synthesize
a
diverse
array
of
other
secondary
metabolites
of
significance
(see
Table
2).
6.
Identifying
fungal
gene
clusters
With
the
availability
of
growing
number
of
fungal
genomes,
there
has
been
a
swift
growth
in
predicting
and
identifying
putative
genes
responsible
for
SM
production.
Bioinformatic
alogorithms
such
as
SMURF
[SM
Unknown
Regions
Finder]
(Khaldi
et
al.,
2010),
antiSMASH
(Medema
et
al.,
2011)
and
FungiFun
(Priebe
et
al.,
2011)
are
available
for
the
identification
of
gene
clusters
responsible
for
secondary
metabolism.
These
tools
help
in
predicting
the
genes
coding
for
core
PKS
and/or
NRPS
enzymes,
along
with
the
pre-
dicted
function
of
the
adjacent
genes
to
aid
in
identification
of
SM
clusters
in
the
genome
(Bergmann
et
al.,
2007).
These
algorithms
have
been
successfully
utilised
to
identify
nearly
50
gene
clusters
in
Aspergillus
sp.
(genome
size
of
28–40
Mb)
and
27
gene
clusters
in
Arthroderma
sp.
(genome
size
of
22
Mb)
(Burmester
et
al.,
2011;
von
Dohren,
2009).
Since
the
structural
and
functional
character-
istics
of
the
metabolites
encoded
by
these
clusters
remain
largely
unknown,
these
are
termed
‘cryptic’
or
‘orphan’
clusters
(Bergmann
et
al.,
2007;
Hertweck,
2009b).
7.
Regulation
of
secondary
metabolism
in
fungi
Various
factors
like
chromosomal
organization
of
SM
genes,
developmental
conditions
and
environmental
signals
play
a
sig-
nificant
role
in
SM
regulation
in
fungi
(Shwab
and
Keller,
2008).
Transcription
factors
also
tightly
regulate
transcription
of
gene
clusters
involved
in
secondary
metabolism.
Several
regulatory
pro-
teins
are
known
to
control
SM
regulation
in
filamentous
fungi
and
these
regulators
can
be
broadly
classified
as
pathway-specific
or
global
regulators
(Table
3).

V.B.
Deepika
et
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/
Microbiological
Research
182
(2016)
125–140
129
Table
3
Examples
of
regulatory
proteins
functioning
via
pathway
specific
regulation.
Regulator
Protein
Class
of
regulatory
protein
Organism
Metabolite
References
MlcR
Zn(II)2Cys6DNA-binding
protein
Penicillium
citrinum
Compactin
Abe
et
al.
(2002)
Tri6
Cys2His2
zinc
finger
proteins
Fusarium
sporotrichioides
Trichothecene
Proctor
et
al.
(1995)
ToxE
Ankyrin
repeat
protein
Cochliobolus
carbonum
HC-Toxin
Pedley
and
Walton
(2001)
CPCR1
2-Peptide
forkhead
protein
Acremonium
chrysogenum
cephalosporin
C
Schmitt
et
al.
(2004a)
AcFKH1
2-Peptide
forkhead
protein
Acremonium
chrysogenum
cephalosporin
C
Schmitt
et
al.
(2004b)
PENR1
HAP-like
transcription
factor
Aspergillus
nidulans
Penicillin
Litzka
et
al.
(1998)
7.1.
G-protein
signaling
in
filamentous
fungi
Heterotrimeric
G-proteins
are
conserved
in
most
eukary-
otes
and
play
an
important
role
in
transmitting
external
cues
into
the
cells
thereby
eliciting
specific
biochemical
and
phys-
iological
responses
(Morris
and
Malbon,
1999).
Studies
on
G
protein–mediated
signaling
in
numerous
filamentous
fungi
led
to
understanding
of
regulation
of
biosynthesis
of
fungal
sec-
ondary
metabolites.
A
seven-transmembrane-spanning
domain
G
protein–coupled
receptor
(GPCR),
a
G
protein
that
consists
of
␣,
␤,
and

subunits,
and
a
secondary
messenger
producing
effec-
tor
involved
in
initiating
and
amplifying
cellular
responses
in
a
sequential
and
coordinated
manner
form
the
structural
unit
of
heterotrimeric
G
protein
signaling
complex
(Morris
and
Malbon,
1999).
GPCR-G
protein
signaling
initiates
two
downstream
cascade
pathways—one
ending
in
protein
kinase
A
(PKA)
through
adenylyl
cyclase
and
cAMP;
the
other
one
being
mitogen-activated
protein
kinase
cascade.
These
in
turn
activate
cellular
responses
such
as
growth,
mating,
cell
division,
cell–cell
fusion,
morphogenesis,
tox-
icogenesis,
chemotaxis
and
pathogenesis
(Bolker,
1998;
Lengeler
et
al.,
2000;
Tilburn
et
al.,
1995).
7.2.
Fungal
development
and
secondary
metabolism
The
close
correlation
between
the
secondary
metabolism
and
fungal
development
has
been
reviewed
by
Calvo
et
al.
(2002).
A
classic
example
illustrating
the
association
between
synthesis
of
a
natural
product
and
morphological
development
(Bennett
and
Ciegler,
1983)
is
seen
in
‘sec’
mutants
of
Aspergillus
parasiticus.
These
mutants
show
lack
of
sporulation
and
reduced
aflatoxin
pro-
duction
(Kale
et
al.,
1994).
It
was
observed
that
even
with
the
presence
of
genes
which
encodes
necessary
enzymes
for
aflatoxin
synthesis
(Kale
et
al.,
1996),
the
toxigenic
strains
showed
5–10
fold
reduction
in
the
expression
of
pathway-specific
regulator,
aflR
as
compared
to
parental
strains
(Kale
et
al.,
2003).
Similarly,
mutation
observed
in
fluP
gene
resulted
in
a
fluffy
hyphal
phenotype
and
reduced
production
of
asexual
spores
and
aflatoxin
(Zhou
et
al.,
2000)
suggesting
interplay
between
secondary
metabolism
and
fungal
growth.
In
the
filamentous
fungus
A.
nidulans,
the
genes
flbA
and
fadA
control
the
shift
from
growth
to
sporulation
and
mycotoxin
(sterig-
matocystin)
production.
FlbA,
a
G
protein
signaling
regulator,
is
required
to
repress
growth
signaling
via
FadA
(␣
subunit
of
het-
erotrimeric
G
protein).
For
the
sporulation
and
sterigmatocystin
(ST)
production,
the
organism
is
dependent
on
FlbA-dependent
inhibition
of
FadA
signaling
(Adams
et
al.,
1998;
Yu
et
al.,
1996a).
Negative
regulation
of
aflatoxin
(AF)
and
ST
synthesis
(Hicks
et
al.,
1997;
Shimizu
and
Keller,
2001)
is
controlled
by
FadA
and
PkaA,
a
protein
kinase
downstream
in
the
pathway
in
A.
nidulans.
Both
tran-
scriptional
and
post-transcriptional
regulation
of
ST
production
is
carried
out
by
PkaA
through
aflR
and
its
gene
product
(Shimizu
and
Keller,
2001).
In
A.
parasiticus,
a
similar
control
of
aflatoxin
synthe-
sis
via
regulatory
cascade
has
been
postulated
(Roze
et
al.,
2004).
Inactivation
of
aflR
is
via
phosphorylation
by
PkaA
(Shimizu
et
al.,
2003)
and
this
is
further
mediated
through
LaeA,
a
master
regu-
lator
with
methyltransferase
activity
(Bok
and
Keller,
2004).
FadA
has
been
found
to
negatively
regulate
AF
and
ST
synthesis,
but
in
A.
nidulans
it
positively
regulates
penicillin
production.
However,
the
homologue
of
FadA
in
Fusarium
sporotrichioides
is
known
to
regulate
trichothene
production
positively
(Tag
et
al.,
2000).
The
regulation
of
secondary
metabolism
via
G
protein
signaling
has
also
been
observed
in
Botrytis
cinerea
and
Trichoderma
atroviride
(Gronover
et
al.,
2004;
Reithner
et
al.,
2005).
Conidiation
and
ST
production
via
the
gene
fluG
is
linked
to
asex-
ual
sporulation
(Lee
and
Adams,
1994,
1995).
FluG
acts
indirectly
on
ST
biosynthesis,
by
activating
flbA
and
thereby
repressing
FadA
sig-
naling
(Seo
et
al.,
2003).
RasA
is
quite
another
GTP-binding
signaling
protein
which
is
involved
in
ST
production,
which
upon
activa-
tion
represses
aflR
expression
thereby
inhibiting
ST
biosynthesis
(Shimizu
et
al.,
2003)
(Fig.
1).
PkaA
partially
controls
the
post-transcriptional
regulation
of
AflR
by
RasA
(Shimizu
et
al.,
2003).
Another
positive
regulator,
PhlP
also
promotes
ST
production
in
A.
nidulans,
further
suggest-
ing
multiple
effects
of
G-protein
signaling
pathway
on
secondary
metabolism
(Seo
and
Yu,
2006).
Pathogenicity
is
also
a
major
process
regulated
by
G-protein
signaling
apart
from
controlling
the
regulation
of
secondary
metabolism
and
spore
formation.
The
importance
of
cpg1,
which
is
responsible
for
encoding
the
␣-
subunit
of
G
protein,
was
clearly
observed
in
the
cpg1mutant
of
Cryphonectria
parasitica.
This
fungus
responsible
for
causing
chest-
nut
blight
disease
showed
visible
reduction
in
growth
rate
along
with
decreased
levels
of
sporulation,
pigmentation
and
reduced
virulence
(Gao
and
Nuss,
1996).
7.3.
Velvet
complex
as
a
regulator
Velvet
complex
is
a
developmental
regulator
conserved
in
most
fungi
except
yeasts.
Development
and
secondary
metabolism
is
linked
by
this
heteromeric
complex
(Bayram
et
al.,
2008;
Bayram
and
Braus,
2011).
LaeA,
VeA
and
VelB
form
the
velvet
complex
of
A.
nidulans
where
LaeA
acts
a
major
link
between
light
regulation
and
chromatin
rearrangement
(Bayram
et
al.,
2008).
Sexual
devel-
opment
in
A.
nidulans
is
regulated
by
a
light-dependent
regulator
VeA.
The
velvet
complex
formed
in
A.
nidulans,
A.
chrysogenum
and
P.
chrysogenum
also
regulates
␤-lactam
biosynthesis
(Dreyer
et
al.,
2007;
Kato
et
al.,
2003;
Kosalkova
et
al.,
2009).
In
Aspergillus
flavus,
VeA
has
been
shown
to
control
the
regulatory
gene
aflR
and
subsequently
aflatoxin
production
(Duran
et
al.,
2007;
Kato
et
al.,
2003).
COP9
signalosome
is
another
protein
complex
that
regulates
the
transition
between
secondary
metabolism
and
hyphal
develop-
ment
(Nahlik
et
al.,
2010).
7.4.
Role
of
ligands
Hormone-like
molecules
such
as
oxylipins
and
conidiogenones
have
been
reported
to
regulate
SM
production
(Roncal
et
al.,
2002;
Tsitsigiannis
and
Keller,
2007).
These
oxygenated
lipid
molecules
synthesized
by
ppo
genes
that
code
for
fatty
acid
oxygenases
reg-
ulate
the
fine
equilibrium
between
sexual
and
asexual
sporulation
in
aspergilli
(Tsitsigiannis
et
al.,
2004).
Deletion
of
ppoA
and
ppoC
genes
resulted
in
decreased
levels
of
ST
production
and
increased
penicillin
production
in
A.
nidulans
further
indicating
an
extra

130
V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
Fig.
1.
Coordinated
regulation
between
fungal
development
and
SM
synthesis.
G-protein
coupled
receptor
gets
activated
via
extracellular
ligand
molecule
and
leads
to
a
cascade
involving
Pka
mediated
regulation
of
SM
synthesis.
␣
+
GTP
(FadA)
has
a
negative
effect
on
AF/ST
synthesis
and
positively
regulates
penicillin
biosynthesis.
PkaA
inactivates
aflR
by
phosphorylation
thereby
preventing
SM
synthesis.
FluG
regulates
between
conidiation
and
SM
synthesis.
Activation
of
FlbA
by
FluG,
inhibits
FadA
activity
thus
posing
positive
effect
on
SM
synthesis.
RasA
is
another
GTP-binding
signaling
protein
involved
in
ST
production,
which
upon
activation
represses
aflR
expression
and
inhibits
ST
biosynthesis
while
PhlP
acts
as
a
positive
regulator
in
promoting
ST
production
in
A.
nidulans.
LaeA
is
a
master
regulator
and
controls
ST
biosynthesis
by
chromatin
modification.
link
between
fungal
morphogenesis
and
secondary
metabolism
(Tsitsigiannis
and
Keller,
2006).
The
inverse
regulation
of
ST
and
penicillin
cluster
regulation
indicates
that
Ppo
proteins
are
further
under
the
control
of
FadA
and
PkaA
(Shwab
and
Keller,
2008).
7.5.
Regulation
through
environmental
stimuli
Fungal
SM
production
may
also
be
influenced
by
external
factors
like
temperature,
nutrient
sources,
pH,
light,
as
well
as
interaction
between
the
host
and
the
fungus.
Signaling
proteins
like
CreA
(car-
bon
signaling),
PacC
(pH
signaling)
and
AreA
(nitrogen
signaling)
(Dowzer
and
Kelly,
1989;
Hynes,
1975;
Tilburn
et
al.,
1995)
are
zinc-
finger
proteins
of
Cys2His2type
(Shwab
and
Keller,
2008)
that
play
a
significant
role
in
metabolite
regulation
by
transmitting
environ-
mental
signals.
CreA
and
PacC
proteins
regulate
penicillin
synthesis
in
a
positive
and
negative
way
respectively
(Martin,
2000).
In
Fusar-
ium
fujikuroi,
the
gibberlin
biosynthetic
gene
cluster
is
under
the
negative
regulation
of
AreA,
a
global
nitrogen
regulator
(Tudzynski
et
al.,
1999).
On
the
contrary,
AreA
regulation
activates
fumonisin
B1
synthesis
in
Fusarium
verticillioides
(Kim
and
Woloshuk,
2008).
The
biosynthesis
of
gibberellins
by
F.
fujikuroi
was
found
to
be
due
to
the
TOR
kinase,
which
regulates
nutrient
mediated
growth
sig-
naling
in
budding
yeast
(Teichert
et
al.,
2006).
Fungi
are
generally
known
to
utilize
glucose
over
any
other
sources
of
carbon
like
lactose
which
is
weakly
metabolized
(Brakhage,
1998).
High
glucose
concentration
however
reduces
cephalosporin
production
partly
mediated
by
repression
of
ipnA
and
cefEF
by
CreA
homologue
Cre1
(Janus
et
al.,
2008;
Jekosch
and
Kuck,
2000).
Sub-optimal
growth
conditions
favor
the
production
of
SM
which
likely
reflects
the
growth
conditions
of
fungi
in
natural
habitats
(Brakhage,
1998).
Competitive
interaction
of
the
microbial
communities
within
the
plant
tissue
may
also
trigger
accumulation
of
natural
products
in
vivo
(Ho
et
al.,
2003;
Pettit,
2009).
Reports
suggest
that
metabolic
profile
of
the
endophyte
also
may
be
altered
according
to
fluctuations
in
the
environment
within
the
symbiotic
continuum
of
the
endophyte–plant
interaction
(Suryanarayanan
et
al.,
2009).
7.6.
Pathway
specific
regulation
Some
of
the
secondary
metabolite
gene
clusters
possess
pathway-specific
regulators
that
have
positive
effect
on
gene
expression.
These
proteins
are
often
Zn2Cys6zinc
binuclear
cluster
proteins
(Proctor
et
al.,
1995;
Woloshuk
et
al.,
1994).
Regulation
mediated
by
pathway-specific
proteins
can
be
broadly
studied
under
three
sections:
7.6.1.
In-cluster
genes
encoding
pathway-specific
regulators
AF
and
ST
are
closely
related
mycotoxins
that
have
its
origin
from
similar
gene
clusters
(Brown
et
al.,
1996;
Yu
et
al.,
2004).
aflR
found
in
AF
clusters
of
A.
flavus
and
A.
parasiticus
and
ST
cluster
of
A.
nidulans
(Fernandes
et
al.,
1998;
Woloshuk
et
al.,
1994;
Yu
et
al.,
1996b)
codes
for
a
Zn(II)2Cys6protein
which
recognizes
and
binds
to
a
palindromic
sequence
TCG(N5)GCA
in
the
AF/ST
gene
promoter
region
(Ehrlich
et
al.,
1999;
Payne
and
Brown,
1998)
(Fig.
2).
In
A.
flavus
and
A.
parasiticus,
a
second
binding
site
with
the
sequence
TTAGGCCTAA
is
considered
to
be
important
in
AflR
autoregulation
(Chang
et
al.,
1995,
1999).
Deletion
of
aflR
leads
to
loss
of
tran-
scription
of
AF/ST
biosynthetic
genes
leading
to
reduction
in
AF/ST
production
(Yu
et
al.,
1996b).
AF/ST
clusters
also
produce
AflJ
lead-
ing
to
optimal
gene
cluster
expression
presumably
by
its
interaction
with
AflR
while
absence
of
aflJ
is
shown
to
result
in
low
levels
of
expression
in
A.
parasiticus
(Chang
et
al.,
2000;
Meyers
et
al.,
1998);
but
its
specific
role
has
not
yet
been
assigned
in
any
other
aspergilli.
In
certain
gene
clusters,
transcriptional
activation
of
member
genes
is
regulated
by
the
transcriptional
regulator
present
within
the
cluster
(Fig.
2);
for
example,
ApdR
regulates
all
the
genes
in
the
aspyridone
biosynthesis
cluster
in
A.
nidulans
(Bergmann
et
al.,
2007).
Gliotoxin
biosynthetic
gene
cluster
consisting
of
13
genes
(Cramer
et
al.,
2006)
is
regulated
by
the
Zn-finger
transcription
factor
GliZ53
which
is
coded
by
one
of
the
genes
in
the
cluster.

V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
131
Fig.
2.
Activation
of
SM
cluster
by
Transcription
factors
(TF)
encoded
by
cluster-
specific
genes.
Zinc
binuclear
cluster
proteins
(Zn2Cys6)
encoded
by
aflR
activates
Aflatoxin
and
Sterigmatocystin
synthesis.
Aspyridone
synthesis
is
activated
by
TF
encoded
by
apdR;
a
Zinc
finger
TF
GliZ53
encoded
by
gliZ
activates
Gliotoxin
syn-
thesis.
Deletion
of
this
gene
led
to
loss
of
gliotoxin
production,
while
overexpression
substantially
increased
the
gliotoxin
levels
(Bok
et
al.,
2006a).
GliZ
is
responsible
for
the
transcription
of
all
genes
except
gliotoxin
oxidase
encoding
gliT,
which
confers
resistance
to
gliotoxin.
Even
though
the
promoter
region
of
all
the
gli
genes
con-
tains
the
GliZ-binding
motifs,
it
was
observed
that
transcription
of
gliT
was
induced
even
in
gliZ
deletion
mutant
when
gliotoxin
is
added
exogenously
to
the
cultures
(Schrettl
et
al.,
2010).
7.6.2.
Position
of
the
regulatory
gene
A
crucial
factor
that
aids
in
gain
or
loss
of
the
pathway-specific
regulator
gene
is
their
positioning
in
relation
to
SM
gene
cluster.
An
additional
copy
of
aflR
in
a
laeA
mutant
strain
plays
an
important
role
in
the
expression
of
ST
biosynthesis
in
A.
nidulans
(Bok
et
al.,
2006b).
In
a
laeA-null
mutant,
both
aflR
and
ST
biosynthesis
gene
stcU
were
expressed
when
an
additional
copy
of
aflR
was
placed
at
the
trpC
locus
but
similar
effect
was
not
observed
when
the
same
gene
was
placed
within
the
ST
cluster
(Fig.
3).
In
yet
another
experiment,
it
was
shown
that
the
argB
(coding
for
ornithine
car-
bamoyltransferase
mitochondrial)
involved
in
primary
metabolism
was
regulated
by
LaeA
when
it
was
placed
under
the
gene
clus-
ter
suggesting
that
relative
positioning
of
genes
in
relation
to
gene
clusters
has
profound
effects
on
its
regulation.
7.6.3.
Cross-talk
between
gene
clusters
A
silent
inp
cluster
consisting
of
inpA
and
inpB
(NRPS
cod-
ing
genes)
and
a
pathway
specific
regulator
scpR
(secondary
metabolism
cross
pathway
regulator)
was
identified
in
A.
nidulans.
Expression
of
this
silent
cluster
was
achieved
by
inducing
scpR
with
the
promoter
of
regulatable
alcohol
dehydrogenase
A
gene
(alcA).
This
also
led
to
expression
of
afo
gene
cluster
involved
in
asperfura-
none
synthesis
suggesting
ScpR
controls
afo
gene
cluster
by
binding
to
the
promoter
of
afo
(Bergmann
et
al.,
2010).
Both
the
inp
and
afo
clusters
remain
silent
in
the
absence
of
ScpR
and
are
even
located
on
two
different
chromosomes
(chromosome
II
and
chromosome
VIII
respectively).
Such
crosstalk
between
regulators
and
fungal
gene
clusters
might
be
the
basis
for
combinatorial
biosynthetic
pathways
resulting
in
even
more
compounds
(Fig.
4).
7.7.
Global
regulation
in
fungi
Globally
acting
transcription
factors
encoded
by
genes
which
are
not
cluster-specific
control
the
global
regulation
and
also
regulate
expression
of
a
number
of
genes
not
connected
to
sec-
ondary
metabolism.
CCAAT-binding
complex
(CBC)
and
PacC,
a
pH
response
transcription
factor
(Brakhage
et
al.,
1999)
are
exam-
ples
of
global
transcription
factors
that
regulate
various
SM
genes
and
especially
clusters
that
lack
pathway-specific
regulatory
genes
(Bomke
and
Tudzynski,
2009)
such
as
gene
clusters
involved
in
␤-lactam
biosynthesis
(Fig.
5).
Environmental
stimuli
that
induce
secondary
metabolite
clus-
ters
are
invariably
associated
with
global
transcription
factors.
PacC,
one
of
the
major
global
regulators
is
also
the
key
deter-
minant
in
responses
to
changes
in
pH
(Tilburn
et
al.,
1995).
In
A.
nidulans,
PacC
is
known
to
regulate
expression
of
several
genes
including
palD
(alkaline
phosphatase
D),
acvA
(N-(5-
amino-5-carboxypentanoyl)-1-cysteinyl-d-valine
synthase)
and
ipnA
(isopenicillin
N
synthase)
in
penicillin
gene
cluster
at
alka-
line
pH
(Bergh
and
Brakhage,
1998;
Espeso
and
Penalva,
1996;
Tilburn
et
al.,
1995).
Interestingly,
PacC
and
its
homologues
seem
to
negatively
regulate
the
ST
biosynthesis
gene
expression
(Keller
et
al.,
1997).
This
pH
mediated
regulation
of
penicillin
biosynthe-
sis
supports
the
finding
that
␤-lactam
antibiotics
exhibit
increased
anti-bacterial
activity
towards
bacterial
species
at
alkaline
pH
(Arst,
1996).
Three
proteins
HapB,
HapC
and
HapE
form
the
CBC
com-
plex
which
is
involved
in
various
physiological
signals
including
signaling
iron
deprivation
and
redox
status
(Hortschansky
et
al.,
2007;
Thon
et
al.,
2010).
The
binding
of
CBC
to
CCAAT
boxes
in
the
promoter
region
of
penicillin
biosynthesis
genes
in
A.
nidu-
lans
leads
to
expression
of
ipnA
and
aatA
(Brakhage
et
al.,
2009a).
Under
iron-deprived
conditions,
iron-scavenging
siderophores
are
synthesized
under
the
influence
of
HapX
protein
that
specifically
binds
to
CBC
complex
(Hortschansky
et
al.,
2007).
The
conserved
cysteine
residues
in
the
HapC
subunit
of
CBC
plays
an
important
role
in
post-translational
regulation
and
nuclear
localization
of
CBC66
and
also
recognizes
the
redox
status
of
the
cell
that
influences
peni-
cillin
biosynthesis
in
P.
chrysogenum
(Cohen
et
al.,
1994).
Yap1
is
Fig.
3.
Strategies
followed
in
activation
of
SM
gene
clusters.
Positioning
of
the
regulatory
gene
in
relation
to
gene
cluster
determines
the
activity.
In
A.
nidulans
incorporating
a
primary
metabolism
gene
argB
into
ST
cluster
leads
to
synthesis
of
ST
which
is
under
the
control
of
LaeA
(A).
In
laeA
mutants,
position
of
additional
aflR
has
a
crucial
role.
Additional
aflR
in
ST
cluster
did
not
lead
to
ST
synthesis
(B)
whereas
aflR
incorporation
in
trpC
locus
activated
the
ST
gene
cluster
(C).

132
V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
Fig.
4.
Diagram
illustrating
the
cross
talk
between
regulatory
elements
and
gene
clusters
in
filamentous
fungi.
Activation
of
ScpR,
a
regulatory
protein
encoded
by
scpR
located
in
inp
cluster
in
chromosome
II,
activates
silent
inpA
and
inpB
genes
along
with
afo
gene
clusters
in
chromosome
VIII
via
activation
of
AfoA
regulatory
protein
in
A.
nidulans.
Fig.
5.
Effect
of
external
factors
on
activation
of
SM
clusters.
Global
regulatory
proteins
are
activated
by
external
factors
like
redox
potential
via
CCAAT
binding
at
the
promoter
region
of
the
gene
clusters
with
common
examples
being
regulation
by
Hap
Complex
(activating
penicillin
clusters
in
A.
nidulans)
and
Yap1
(activating
aflatoxin
clusters
in
A.
parasiticus).
pH
mediated
regulation
is
activated
by
PacC
in
A.
nidulans
leading
to
production
of
several
essential
enzymes
while
conditions
of
iron
deficiency
leads
to
recruitment
of
HapX
which
binds
to
Hap
complex
and
activates
synthesis
of
siderophores.
yet
another
homologue
involved
in
redox-regulated
pathway
in
A.
parasiticus
that
senses
oxidative
stress
to
activate
aflatoxin
biosyn-
thesis
(Reverberi
et
al.,
2008).
7.8.
Chromatin
structure
and
histone
modifications
in
regulation
Chromatin
forms
a
basic
substrate
for
the
transcriptional
machinery
and
regulatory
proteins
in
the
nucleus.
In
filamentous
fungi,
chromatin
histone
modifications
and
structural
transitions
have
been
studied
mainly
in
the
context
of
DNA
methylation
(Foss
et
al.,
1993;
Kouzminova
and
Selker,
2001;
Tamaru
and
Selker,
2001),
light
regulation
(Grimaldi
et
al.,
2006),
cell
cycle
(Osmani
et
al.,
1991)
and
nitrogen
regulation
(Berger
et
al.,
2006;
Berger
et
al.,
2008;
Reyes-Dominguez
et
al.,
2008).
7.8.1.
Chromatin-mediated
regulation
Histones
H3
and
H4
are
major
scaffold
proteins
in
nucleosome
formation
and
are
also
major
substrates
for
various
modification
events,
including
methylation,
acetylation,
phosphorylation,
and
other
such
modifications
(Shilatifard,
2006).
These
chromatin
mod-
ification
events
lead
to
activation
or
silencing
of
genes
and
have
a
major
advantage
in
regulation
of
individual
SM
gene
clusters
due
to
its
confinement
to
specific
regions
in
the
chromosome.
SM
gene
clusters
are
not
evenly
distributed
within
the
chromosome
but
are
localized
in
sub-telomeric
regions
which
are
highly
prone
to
non-allelic
recombination,
DNA
inversion,
partial
deletions,
translocations
and
other
rearrangements
making
these
regions
hotspots
for
adaptive
evolution.
Four
genes
(acvA,
ipnA,
aatA,
and
aatB)
are
required
for
penicillin
biosynthesis
and
of
these
the
first
three
are
localized
in
Chromosome
VI
as
a
cluster
while
the
fourth
gene
is
present
in
chromosome
I
separately.
Chromosome
VI
is
con-
trolled
by
GcnE
(core
subunit
of
SAGA/ADA
complex
in
A.
nidulans)
histone
acetylase
while
aatB
located
in
another
chromosome
away
from
this
cluster
is
not
under
GcnE
regulation.
Nutzmann
et
al.
(2011)
found
that
soil-dwelling
bacterium
Streptomyces
could
acti-
vate
silent
gene
clusters
in
A.
nidulans
by
histone
modification
via
SAGA/ADA
complex
but
the
histone
acetylation
was
found
to
be
cluster
specific
and
had
no
effect
on
aatB
expression.
7.8.2.
Histone
methylation
Keller
et
al.
(2005)
conducted
complementation
studies
on
ST
biosynthetic
mutants
that
led
to
the
identification
of
LaeA,
which
shows
sequence
similarity
to
arginine
methyltransferases
and
histones,
as
a
chromatin
remodeling
agent.
Microarray
anal-
ysis
results
of
A.
nidulans
laeA
mutants
show
down-regulation
of
ST
and
penicillin
biosynthesis
gene
clusters
and
some
indole
alka-
loid
biosynthesis
genes
(Yin
and
Keller,
2011).
A
complete
genome
analysis
conducted
on
transcriptional
profiles
on
three
A.
fumiga-
tus
strains
(wild-type,
laeA
mutant
and
complemented
control),
showed
that
59%
of
the
analyzed
SM
gene
clusters
(13
of
22
genes)
were
expressed
at
much
lower
levels
in
laeA
mutant.
Studies
indi-
cate
that
9.5%
of
all
the
genes
as
well
as
nearly
40%
of
key
SM
genes
in
A.
fumigatus
are
under
the
influence
of
LaeA
(Perrin
et
al.,
2007).
Studies
on
other
histone-modifying
enzymes
also
provide
further
evidence
that
LaeA
mediated
changes
are
due
to
chromatin
modi-
fication.
Increased
heterochromatin
protein
1
(HepA)
marks
on
the

V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
133
Fig.
6.
Histone
methylation
and
increased
HepA
binding
at
SM
clusters
during
the
active
growth
phase
of
the
fungus
inhibits
ST
synthesis.
Counteracting
effect
of
acetylation
and
decreased
levels
of
HepA
upon
growth
arrest
initiates
ST
synthesis.
Inhibitor
of
methylation
(LaeA)
and
protein
complexes
like
Compass,
SAGA-ADA
with
acetyltransferase
activity
act
as
activator
of
SM
synthesis
while
HdaA
with
deacetylase
activity
exhibits
negative
regulation
towards
SM
synthesis.
ST
biosynthesis
gene
cluster
by
histone
methylation
(H3K9me3
and
H3K4me)
are
observed
during
active
growth
phase
(Fig.
6).
Upon
arrested
growth
and
activation
of
ST
production,
there
is
a
decrease
in
the
levels
of
HepA
and
H3K9me3
with
concomitant
increase
in
the
H3
acetylation
levels
at
ST
biosynthesis
gene
clus-
ters.
laeA
deletion
drastically
increases
the
levels
of
HepA
binding
at
aflR
promoter
(Fig.
6)
indicating
the
counteractive
effect
of
LaeA
on
heterochromatic
marks
(Reyes-Dominguez
et
al.,
2010).
A
novel
protein
RsmA
(restorer
of
secondary
metabolism
A)
compensates
for
the
loss
of
LaeA
and
autonomously
enhances
SM
production
in
A.
nidulans
in
the
presence
of
velvet
complex
(Shaaban
et
al.,
2010).
In
addition,
RsmA
overexpression
greatly
increased
ST
production
by
binding
to
aflR
promoter
at
two
sites.
Compass
(complex
proteins
associated
with
Set1)
complex
con-
trols
H3K4me
in
Saccharomyces
cerevisiae
(Mueller
et
al.,
2006).
In
A.
nidulans,
cclA,
a
known
ortholog
of
S.
cerevisiae
BRE2,
codes
for
one
of
the
eight
compass
proteins.
Two
silent
gene
clusters
that
code
for
monodictyphenone
and
emodins
(active
anthraquinone
con-
stituents)
and
anti-osteoporosis
polyketides
were
activated
in
cclA
deletion
mutants
suggesting
its
role
in
silencing
of
gene
clusters
(Bok
et
al.,
2009).
7.8.3.
Histone
acetylation—an
essential
mark
for
SM
gene
activation
Activation
of
genes
involves
acetylation
of
histones
through
multi-subunit
biological
complexes
like
Saga/Ada
or
NuA4
hav-
ing
histone
acetyltransferase
(HAT)
activity
(Brownell
et
al.,
1996;
Grant
et
al.,
1997,
1998)
though
the
molecular
function
of
these
complexes
in
filamentous
fungi
is
sparsely
known.
Elicitation
of
conidiation
(Reyes-Dominguez
et
al.,
2008)
and
nitrogen
assimila-
tion
regulation
(Berger
et
al.,
2008;
Reyes-Dominguez
et
al.,
2008)
in
A.
nidulans
requires
the
regulatory
and
catalytic
subunits
of
SAGA/ADA,
GcnE,
and
AdaB.
Secondary
metabolite
production
led
to
increased
acetylation
of
H3K9
and
H3K14
at
the
ST
locus
of
A.
nidulans
as
confirmed
by
Chromatin
immunoprecipitation
(ChIP)
techniques
(Reyes-Dominguez
et
al.,
2010).
Nutzmann
et
al.
(2011)
confirmed
the
need
of
catalytic
and
regulatory
subunits
of
GcnE
and
AdaB
for
increase
in
H3
acetylation
and
showed
that
gene
clus-
ters
of
penicillin,
terrequinone
and
orsellinic
acid
are
also
under
the
regulation
of
the
SAGA/ADA
co-activator
complex.
Addition
of
HDAC
inhibitor
suberoylanilide
hydroxamic
acid
(SAHA)
induces
orsellinic
acid
cluster
while
addition
of
HAT
inhibitor
anacardic
acid
blocks
the
expression
(Nutzmann
et
al.,
2011).
Spiroan-
thrones,
sanghaspirodins
A
and
B
and
novel
anti-proliferative
compounds
were
obtained
under
severe
nitrogen
limitation
con-
ditions
(Scherlach
et
al.,
2011)
suggesting
a
major
role
for
AreA,
a
transcriptional
co-activator
(Berger
et
al.,
2008;
Scazzocchio
2000;
Schinko
et
al.,
2010),
in
orsellinic
acid
cluster
activation.
Studies
in
A.
nidulans
also
suggest
that
activation
of
silent
clusters
by
bac-
terial
fungal
co-cultivation
were
due
to
increased
level
of
histone
acetylation
at
SM
clusters.
Stress
imposed
on
fungi
by
bacterial
cells
can
activate
gene
clusters
since
the
oxidative
stress
pathway
medi-
ated
by
cAMP-response
elements
is
involved
in
the
activation
of
the
aflatoxin
gene
cluster
in
A.
parasiticus
(Roze
et
al.,
2011a).
Soukup
et
al.
(2012)
examined
four
gene
clusters
for
H4
acetylation
marks
(sterigmatocystin,
penicillin,
terrequinone,
and
orsellinic
acid)
and
found
increased
H4K12
acetylation
in
tested
clusters
during
SM
production.
Suppression
and
over
expres-
sion
studies
indicate
a
major
role
for
histone
acetyltransferase
EsaA
in
SM
expression
and
establishment
of
specific
chromatin
marks.
Esa1
is
the
catalytic
subunit
of
NuA4
transcriptional
adap-
tor/acetyltransferase
complex
involved
in
H2A
and
H4
acetylation,
cell
cycle
control,
and
epigenetic
control
of
transcription
in
S.
cere-
visiae
while
EsaA
in
A.
nidulans
was
found
to
be
the
ortholog
of
Eas1
(Allard
et
al.,
1999;
Doyon
and
Cote,
2004;
Galarneau
et
al.,
2000).
LaeA
plays
a
major
role
in
H4
acetylation
by
EsaA
suggest-
ing
that
this
general
activator
of
SM
and
member
of
the
velvet
complex
(Bayram
et
al.,
2010;
Bayram
and
Braus,
2011)
partici-
pates
in
recruitment
of
the
putative
A.
nidulans
NuA4
complex.
Both
SAGA/ADA-mediated
H3
and
NuA4-mediated
H4
acetylation
seem
to
be
essential
for
activation
of
several
SM
gene
clusters.
8.
Modifications
to
overcome
metabolic
toxicity
Hosts
such
as
Taxus
brevifolia
(paclitaxel),
Catharanthus
roseus
(vinblastine
and
vincristine),
Colchicum
autumnale
(colchicine)
and
Podophyllum
spp.
(Podophyllotoxin)
that
synthesize
antimitotic
agents
provide
specific
clues
towards
possibility
of
develop-
ment
of
self-resistance
mechanism
through
adaptive
coevolution
(Sirikantaramas
et
al.,
2008).
Resistance
in
endophytes
to
over-
come
metabolite
toxicity
however
may
have
been
a
result
of
stable
coexistence
with
host
(Kusari
et
al.,
2012b).
Endophytes
have
also
evolved
different
mechanisms
to
overcome/alleviate
the
effects
of
production
of
metabolites
that
may
be
toxic.
Kusari
et
al.
(2011)
suggested
that
endophytic
Fusarium
solani
in
Camptotheca
over-
come
inhibition
by
camptothecin
(CPT)
by
altering
specific
amino
acid
residues
in
the
Topoisomerase
1.
Another
endophyte,
Albonec-
tria
rigidiuscula,
in
spite
of
not
synthesizing
CPT
showed
extremely
similar
Topo1,
thereby
conferring
CPT
resistance.
This
also
suggests
that
those
fungi
evolved
to
have
properties
conferring
CPT
resis-
tance
are
primed
to
invade
and
sustain
as
endophytes
in
the
host.
While
CPT
producing
Camptotheca
acuminata,
Ophiorrhiza
pumila
and
Ophiorrhiza
liukiuensis
are
shown
to
harbor
topoisomerase
I
with
point
mutations,
Ophiorrhiza
japonica
which
is
not
known
to
produce
CPT
exhibits
partial
resistance
to
camptothecin
in
vivo
(Sirikantaramas
et
al.,
2008).
This
suggests
the
possible
mechanism
of
pre-adaptation
towards
topoisomerase
I
resistance
in
O.
japon-
ica.
The
above
studies
point
to
a
long
time
evolutionary
exposure

134
V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
Fig.
7.
Mechanisms
involved
in
gene
regulation
for
SM
synthesis
in
fungi.
(A).
Activation
of
SM
clusters
by
environmental
stimulus
via
transcription
factors
PacC
(pH
regulation),
AreA
(nitrogen
signaling),
CreA
(carbon
signalling)
and
LaeA,
VeA
and
VelB
(involved
in
formation
of
velvet
complex
in
light-dependent
signaling)
(B).
Chromatin
modification
due
to
transcription
proteins
like
LaeA.
(C)
Microbial
interactions
leading
to
release
of
unknown
effector
molecules
that
activate
silent
clusters.
(D).
Over-
expression
of
apdR
gene
under
alcA
promoter
(P-alcA)
leading
to
formation
of
new
metabolites
(E).
Gene
disruptions
[easB
(in
PKS
pathway)
and
easA
(in
NRPS
pathway)]
leading
to
production
of
emericellamides.
(F).
Promoter
replacement
with
a
strong
promoter
(P-alcA)
inducing
synthesis
of
asperfuranone.
of
plants
to
CPT
and
suggest
distinct
possibilities
of
co-evolution
of
plant-microbe
association.
9.
Microfungi
as
alternatives
for
plant
bioactive
compound
synthesis
Despite
the
current
knowledge
on
fungal
secondary
metabolism,
certain
limitations
continue
to
hinder
the
potential
of
exploiting
these
fungi
as
alternative
candidates
for
production
of
various
pharmaceutically-important
plant
based
metabolites
(Priti
et
al.,
2009).
Upon
repeated
sub-culturing
of
endophytic
fungi
in
axenic
medium,
they
exhibit
a
typical
tendency
to
lose
their
ability
to
produce
SMs.
This
process
of
‘attenuation’
is
a
common
trend
observed
in
many
fungi,
bacteria
and
viruses.
Various
hypotheses
have
been
put
forth
to
explain
this
process
of
attenuation
in
endophytic
fungi
and
some
of
these
include:
1.
Absence
of
host
specific
stimuli
when
the
fungi
are
cultured
in
axenic
medium,
2.
Gene
silencing
in
axenic
cultures
(Priti
et
al.,
2009),
3.
Loss
of
critical
elements
in
gene
clusters
responsible
for
metabo-
lite
synthesis
in
extra
chromosomal
elements
during
repeated
sub-culturing
and,
4.
Plasmid
curing
from
certain
endohyphal
bacteria
residing
within
endophytic
fungi
(Kumara
et
al.,
2014).
10.
Activation
of
orphan
biosynthetic
gene
clusters
Several
biosynthetic
gene
clusters
remain
turned
off
under
nor-
mal
laboratory
cultivation
conditions
and
hence
there
is
significant
underestimation
of
the
true
metabolic
potential
of
microfungi.
The
availability
of
sequenced
fungal
genomes
and
novel
bioinformatic
approaches
can
lead
to
discovery
of
many
cryptic
natural
com-
pounds
(Brakhage
et
al.,
2009b;
Chiang
et
al.,
2009a;
Hertweck,
2009b).
Genome
mining
of
Aspergillus
nidulans
genome
sequence
identified
53
putative
SM
gene
clusters
(von
Dohren,
2009).
It
still
remains
a
major
challenge
to
comprehend
the
physiological
role
of
these
fungal
gene
clusters
especially
under
controlled
axenic
conditions
(Brakhage
et
al.,
2008).
Microbial
interactions
in
eco-
logical
niche
play
an
important
role
in
obtaining
novel
compounds
from
host
(Netzker
et
al.,
2015)
and
can
also
lead
to
activation
of
silenced
genes.
In
a
co-culture
experiment
conducted
with
a
col-
lection
of
58
actinomycetes,
only
Streptomyces
rapamycinicus
was
reported
to
selectively
activate
the
expression
of
silent
orsellinic
acid
synthase
gene
cluster
in
A.
nidulans
(Schroeckh
et
al.,
2009).
A
silent
polyketide
biosynthesis
gene
cluster
was
activated
due
to
the
close
communication
of
Aspergillus
nidulans
with
a
soil-borne
bacterium
Streptomyces
hygroscopicus.
The
expression
of
emericel-
lamide
biosynthesis
gene
clusters,
first
identified
in
A.
nidulans,
was
enhanced
up
to
100-fold
by
co-cultivation
of
marine
dwelling
Salin-
ispora
arenicola
and
Emericella
sp.
(Chiang
et
al.,
2008;
Oh
et
al.,
2007).
Co-culturing
a
marine
fungus,
Pestalotia
sp.
with
a
marine
Gram-negative
bacterium
Thalassopia
sp.
resulted
in
production
of
Pestalone,
a
potent
antibiotic
against
methicillin-resistant
Staphy-
lococcus
aureus
and
vancomycin-resistant
Enterococcus
faecium
(Cueto
et
al.,
2001).
Similarly,
co-culturing
of
Fusarium
tricinc-
tum
with
Gram-positive
Bacillus
subtilis
elicited
production
of
macrocarpon
C,
2-(carboxymethylamino)
benzoic
acid
and
cit-
reoisocoumarinolin
in
cultures
(Ola
et
al.,
2013).
It
is
believed
that
biosynthesis
of
SM
confers
a
biological
ben-
efit
for
the
producer
in
a
specific
ecological
niche
(Brakhage
et
al.,
2009a;
Firn
and
Jones,
2000).
Significant
changes
in
metabolome

V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
135
can
be
brought
about
by
stress
conditions,
changes
in
culture
con-
ditions
and
epigenetic
modulation
(Fig.
7).
Debbab
et
al.
(2013)
suggested
that
the
vast
array
of
novel
metabolites
synthesized
by
mangrove
endophytes
could
be
attributed
to
stimulation
of
silenced
genes
by
the
stress
imparted
on
these
fungi
due
to
the
extreme
and
unusual
conditions
they
inhabit.
Studies
on
intri-
cate
regulatory
circuits
taking
part
in
pathway
regulation
and
their
effect
on
silencing
gene
clusters
will
provide
information
on
successfully
exploiting
microfungi
for
their
metabolic
diver-
sity
(Brakhage
et
al.,
2009b;
Scherlach
and
Hertweck,
2006,
2009).
Newer
approaches
including
unique
cultivation-dependent
and
molecular
cultivation-independent
methods
have
opened
avenues
for
new
age
of
product
revival
from
soil
microorganisms
and
also
an
opportunity
to
discover
novel
and
potentially
bioactive
entities
(Bergmann
et
al.,
2010).
11.
Current
approaches
in
activation
of
gene
clusters
The
enormous
plunge
in
whole
genome
sequencing
costs
has
resulted
in
sequencing
of
number
of
fungal
genomes
that
are
available
in
public
domain.
The
annotation
of
these
data
has
unearthed
huge
number
of
cryptic
gene
clusters
with
tremendous
potential
(Gross
et
al.,
2007).
Several
strategies
including
genetic
modulations
have
been
proposed
to
utilize
this
huge
reservoir
of
metabolites
for
induction
of
these
cryptic
genes
to
make
it
accessi-
ble
for
the
welfare
of
mankind.
11.1.
Gene
deletions
A
“classical”
strategy
is
to
compare
the
metabolic
profile
of
the
deletion
mutant
with
that
of
the
wild
type
using
HPLC
or
LC–MS
to
identify
novel
metabolites
(Brakhage
and
Schroeckh,
2011).
Chemi-
cal
profiling
of
single
gene
deletion
mutants
in
A.
nidulans
has
led
to
characterization
of
nearly
25
SM
synthases/synthetases
(Andersen
et
al.,
2013).
Chiang
et
al.
(2008)
identified
emericellamide
biosyn-
thesis
gene
cluster
in
A.
nidulans
and
consequently
elucidated
its
biosynthesis
pathway
involving
both
NRPS
and
PKS.
Deletion
stud-
ies
have
also
established
the
role
of
LaeA
(Bok
and
Keller,
2004)
and
VeA
(light-regulated
developmental
factor),
core
components
of
nuclear
complex
(Bayram
et
al.,
2008)
in
SM
synthesis.
A
major
drawback
of
this
technique
is
that
it
could
be
applied
only
to
rela-
tively
fewer
metabolites
that
remain
‘turned
on’
under
laboratory
conditions
and
not
for
those
gene
clusters
that
remain
silent
under
cultured
conditions.
Genome
annotation
studies
have
led
to
discov-
ery
of
nearly
30-40
SM
gene
clusters
per
species
in
aspergilli.
Other
techniques
include
over-expression
of
pathway-specific
gene
regu-
lators
that
are
mostly
present
in
all
SM
gene
clusters.
This
strategy
was
found
to
be
feasible
as
SM
genes
are
usually
clustered
and
controlled
by
a
single
regulator.
In
spite
of
difficulties
involved
in
post-translational
modifications
of
cluster-specific
regulators,
a
pathway-specific
regulatory
gene
apdR
encoding
for
Zn2Cys6
transcription
factor
was
over-expressed
in
A.
nidulans
leading
to
discovery
of
two
novel
metabolites
aspyridones
A
and
B
(Bergmann
et
al.,
2007).
Promoter
exchange
or
substitution
of
putative
NRPS
or
PKS
gene
with
a
strong
regulatable
promoter
resulted
in
synthesis
of
asperfuranone
(Chiang
et
al.,
2009b).
11.2.
Modulation
in
epigenetic
mechanisms
Chromatin-modulating
agents
are
also
known
to
cause
induc-
tion
of
cryptic
fungal
gene
clusters
in
fungi.
Studies
indicate
LaeA
to
be
a
master
regulator
involved
in
biosynthetic
cluster
activation
by
chromatin
remodification
(Keller
et
al.,
2005).
Other
chemical
epigenetic
modifiers
that
inhibit
HDAC
(Histone
deacetylases)
or
DNMT
(DNA
methyltransferases)
have
also
been
shown
to
activate
silenced
gene
clusters.
Williams
et
al.
(2008)
reported
produc-
tion
of
new
cladochromes
and
calphostin
B
by
addition
of
SAHA
(suberoylanilide
hydoxamic
acid),
a
HDAC
inhibitor
in
Cladospo-
rium
cladosporioides
and
de
novo
synthesis
of
several
oxylipins
and
lunalides
by
addition
of
DNMT
inhibitor
5-azacytidine
in
Diatrype
sp..
Henrikson
et
al.
(2009)
demonstrated
SAHA
can
elicit
produc-
tion
of
nygerone
A
in
A.
niger
cultures.
Similarly,
disruption
of
hda
gene
coding
for
HDAC
in
A.
nidulans
led
to
transcriptional
activa-
tion
of
ST
and
penicillin
biosynthesis
clusters
(Shwab
et
al.,
2007).
Recently,
Vasanthakumari
et
al.
(2015)
reported
successful
restora-
tion
of
production
of
camptothecin
in
an
attenuated
endophytic
fungus
by
DNMT
inhibitor
5-azacytidine
treatment.
11.3.
Proteomic
approach
Proteome
analysis
has
also
aided
in
discovering
natural
products
and
their
respective
pathways
(Bumpus
et
al.,
2009).
Pro-
teomic
Investigation
of
Secondary
Metabolism
(PrISM),
a
mass
spectrometry-based
method,
has
been
used
to
search
for
new
metabolites
in
Bacillus
and
Streptomyces
by
synchronized
detection
of
polyketides
and
peptides
encoded
by
PKS
and
NRPS
respectively
(Brakhage
and
Schroeckh,
2011).
11.4.
Genome
mining
Genome
mining
has
become
a
promising
tool
towards
discovery
of
natural
products
and
understanding
of
cryptic
biosynthetic
clus-
ters
due
to
wealth
of
information
coming
from
genome
sequence
analysis
data.
Using
bioinformatics
tools
and
changes
in
cul-
ture
parameters,
Scherlach
and
Hertweck
(2006)
discovered
novel
metabolites
aspoquinolones
A–D.
Bioinformatics
tools
have
also
broadened
the
horizons
to
allow
accurate
prediction
of
key
play-
ers
in
pathways
involving
PKS
or
NRPS
as
well
as
understanding
of
the
target
substrate
and
physicochemical
properties
of
the
end
product
(Brakhage
and
Schroeckh,
2011).
A
genomisotopic
approach
combining
bioinformatic
analysis
of
genome
sequences
and
isotope-guided
fractionation
was
used
to
identify
potential
end
products
of
orphan
gene
clusters
(Gross
et
al.,
2007).
Zerikly
and
Challis
(2009)
suggested
in
vitro
reconstitution
approach
for
prediction
of
substrates
in
cryptic
biosynthetic
pathways
by
incu-
bating
recombinant
biosynthetic
enzymes
from
gene
clusters
with
predicted
substrates
to
identify
protein
products.
These
methods
involve
less
genetic
manipulation
and
aids
in
differentiating
pre-
dicted
SMs
from
those
compounds
bearing
similar
physiochemical
properties.
12.
Heterologous
hosts
for
cloning
and
expression
of
fungal
metabolites
The
major
setback
observed
in
obtaining
important
fungal
polyketides
of
pharmacological
interest
is
that
more
often
the
events
involved
in
their
synthesis
are
only
under
natural
condi-
tions
and
are
difficult
to
obtain
when
cultured
in
axenic
medium.
This
is
due
to
the
filamentous
growth
leading
to
dense
clumps
causing
increased
viscosity
and
less
oxygen
solubility
(Rugbjerg
et
al.,
2013).
Heterologous
hosts
such
as
Saccharomyces
cerevisiae
(Tsunematsu
et
al.,
2013),
Aspergillus
oryzae
and
A.
nidulans
have
been
studied
for
heterologous
expression
of
SM
genes
(Anyaogu
and
Mortensen,
2015).
Development
of
finer
genetic
methods
for
strain
construction
in
S.
cerevisiae
has
led
to
engineering
some
of
SM
clusters
into
chromosomes
or
multi-copy
expression
systems
like
plasmids.
S.
cerevisiae
acts
as
an
apt
source
for
gene
targeting
and
gene
fusion
by
homologous
recombination
since
it
has
a
trivial
endogenous
SM
(Siddiqui
et
al.,
2012).
This
helps
in
the
analysis
of
novel
path-
ways
and
their
respective
end
products
as
the
chances
of
undesired

136
V.B.
Deepika
et
al.
/
Microbiological
Research
182
(2016)
125–140
cross
chemical
reactions
between
the
novel
pathway
under
study
and
yeast’s
endogenous
pathways
are
avoided.
Conversely,
this
also
indicates
that
yeast
is
not
completely
equipped
for
the
SM
synthe-
sis
and
may
lack
the
appropriate
machinery
(Kealey
et
al.,
1998;
Mutka
et
al.,
2006).
A.
parasiticus
is
known
to
harbor
important
enzymes
for
aflatoxin
SM
synthesis
in
vesicles
which
might
not
be
present
in
yeast
(Roze
et
al.,
2011b).
The
low
numbers
of
introns
in
S.
cerevisiae
(Spingola
et
al.,
1999)
as
compared
to
filamentous
fungi
(Kupfer
et
al.,
2004)
may
result
in
differences
in
mRNA
splic-
ing.
Filamentous
fungi
like
A.
oryzae
or
A.
nidulans
are
considered
as
candidate
host
systems
for
heterologous
expression
due
to
their
limited
endogenous
SM
and
availability
of
strong
genetic
manip-
ulations
methods
respectively
(Meyer,
2008;
Meyer
et
al.,
2011).
Improvements
in
genetic
manipulation
tools
has
resulted
in
reduc-
tion
of
accidental
gene
integration
by
mutating
genes
involved
in
non-homologous
end-joining
(Nayak
et
al.,
2006;
Ninomiya
et
al.,
2004);
this
has
raised
the
expectations
towards
using
them
as
hosts
using
reconstitution
approach
for
large-scale
production.
One
of
the
successful
heterologous
expression
in
yeast
includes
synthesis
of
6-
methylsalicylic
acid
(6-MSA)
encoded
by
6-
MSA
synthase
gene
from
Penicillium
patulum
(Kealey
et
al.,
1998;
Wattanachaisaereekul
et
al.,
2008),
synthesis
of
(R)-monocillin
II
via
rdc1/
rdc5
from
Pochonia
chlamydosporia
(Zhou
et
al.,
2010b),
and
hypothemycin
from
hpm3/hpm8
(Zhou
et
al.,
2012).
Sakai
et
al.
(2008)
were
successful
in
cloning
citrinin
biosynthetic
gene
clus-
ter
from
Monascus
purpureus
into
Aspergillus
oryzae
using
a
shuttle
cosmid
vector.
For
successful
activation
of
gene
clusters
in
the
cloning
host,
global
regulator
of
SM
synthesis
LaeA
was
regulated
by
constitutively
active
phosphoglycerate
kinase
promoter.
Two
het-
erologous
gene
clusters
namely
the
monacolin
K
gene
cluster
from
Monascus
pilosus
and
the
terrequinone
A
cluster
from
A.
nidulans
were
cloned
and
successfully
expressed
in
A.
oryzae
(Sakai
et
al.,
2012).
13.
Conclusion
Recent
advances
in
plant
metabolic
engineering
techniques
have
unraveled
new
methods
to
overcome
limitations
attached
with
large-scale
synthesis
of
important
metabolites.
The
promise
of
obtaining
bioactive
molecules
from
cryptic
fungi
including
endo-
phytes
has
opened
up
an
exciting
area
of
research
towards
using
them
as
a
feasible
and
non-controversial
alternative
source
of
important
phytochemicals.
Viruses,
parasites,
bacteria
and
fungi
have
evolved
different
resistance
mechanisms
to
effectively
over-
come
and
negate
the
targets
of
antimicrobial
drugs
currently
available
in
combating
infections,
triggering
an
impending
global
crisis.
The
apparent
need
of
the
hour
is
to
develop,
synthesize
or
discover
new
potent
drug
molecules
that
are
safe
and
efficacious
against
these
multidrug
resistant
microbes.
Synthetic
chemistry
has
not
been
able
to
provide
reliable
and
long-lasting
drugs
and
it
is
indeed
safe
to
assume
that
natural
products
modified
using
combinatorial
chemistry
approach
would
be
a
safe
bet
in
com-
bating
antimicrobial
resistance
(Aly
et
al.,
2011).
Studies
focusing
on
underlying
mechanism
that
regulate
attenuation
would
pro-
vide
much
needed
impetus
on
ensuring
continued
production
of
bioactive
molecules
from
a
veritable
source.
The
tremendous
improvement
in
sequencing
techniques
would
immensely
help
us
to
understand
not
only
the
genes
involved
in
SM
synthe-
sis
but
means
to
regulate
these
gene
clusters.
Combinatorial
multi-disciplinary
approaches
involving
bioinformatics,
molecu-
lar
biology
and
metabolomics
are
needed
to
unravel
the
complex
symbiotic
interactions
between
host
and
microfungi
and
other
co-habiting
partners
to
fully
appreciate
their
true
biosynthetic
potential.
Acknowledgements
TSM
thanks
DST-SERB
for
FastTrack
Scheme
and
Manipal
Uni-
versity
for
support
and
funding.
DVB
thanks
Manipal
University
for
student
fellowship.
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