Simulation of complex natural environmental
conditions has been shown to allow growth of
“unculturable” organisms [e.g., iChip device
(Nichols et al. 2010)] and to mine and isolate
their BGCs.
II. Molecular Biology Methods for
Fungal Natural Product Discovery
A. Manipulation of Regulatory Elements
1. Global Regulators of Fungal Secondary
Metabolism
Secondary metabolism in filamentous fungi, as
well as in actinobacteria, is often associated
with developmental maturity and a transition
to a generative (sporulating) growth mode
(Calvo et al. 2002; Wang 2003). It is therefore
unsurprising that the master regulators that
have a profound impact on the SM in filamentous fungi act pleiotropically and often have an
effect on fungal development and/or stress
response mechanisms (Garcı ´a-Estrada et al.
2018).
It is important to note that while the term ‘global’ is
conventionally used to describe elements with a regulatory effect that is not limited to a single cluster, no
regulator has been described so far that controls all,
rather than a fraction, of SM pathways in a given
organism.
Beginning with the observation that veA, a
gene previously known for its role in the control of the light-dependent morphological
switch between sexual and asexual development in Aspergillus nidulans, has an impact
on the biosynthesis of sterigmatocystin and
penicillin (Kato et al. 2003), researchers in the
field have established a direct link between
morphology, primary metabolism, and the
regulatory response in secondary metabolism.
The velvet domain-containing family of
proteins is conserved within the fungal kingdom (Bayram and Braus 2012). The members of
this family, namely, veA and velB, interact with
a methyltransferase domain-containing nuclear
protein laeA, which was initially found to be
essential for the production of sterigmatocystin
(carcinogen), and penicillin (antibiotic) (Bok
and Keller 2004). Since the discovery of laeA,
this regulatory element has been characterized
in other fungi, where its deletion led to the loss
of production of SMs, such as gliotoxin in
Aspergillus fumigatus (Keller et al. 2006), the
antihypercholesterolemic lovastatin in Aspergillus terreus, as well as aflatoxin and aflatrem
in Aspergillus flavus (Bok et al. 2006a). The laeA
regulome investigation helped correctly identify the biosynthetic gene cluster of the antitumor compound terrequinone A, previously
undescribed in A. nidulans (Bok et al. 2006b;
Bouhired et al. 2007). Similarly, Hong and coworkers identified Aspergillus fumisynnematus
to be a producer of cyclopiazonic acid upon
overexpression of laeA (Hong et al. 2015),
underlining the potential of manipulation of
this global regulator for the purpose of discovery of novel metabolites even in strains with no
readily available genome sequence. For a further overview of research focused on the laeA
regulation of SM, the reader is directed to Yin
and Keller (2011).
Another interesting discovery was that of a
transcriptional regulator designated mcrA
(multicluster regulator) in Aspergillus nidulans
(Oakley et al. 2017). The authors used a mutagenic approach to find genes that trans-activated the backbone enzymes of biosynthetic
gene clusters, which led to the discovery of a
transcription factor with a global repressive
effect on secondary metabolism. When deleted,
the mutant strain not only shows a different
profile of known SMs from the parental strain
but also produces two new cichorine derivatives, along with trace amounts of felinone A,
an antibiotic compound recently discovered in
Beauveria felina (Du et al. 2014). It has also
been shown that mcrA works independently of
laeA, as its deletion stimulates SM biosynthesis
regardless of the presence of laeA; furthermore,
new masses were observed when laeA was overexpressed in a mcrA deletion background.
Expectedly, the deletion of mcrA orthologues
in other fungal strains, namely, A. terreus and
Penicillium canescens, also led to altered metabolic profiles in the nascent mutants (Oakley
et al. 2017). Given the high level of conservation
of mcrA among ascomycetes, it is likely that the
11 New Avenues Toward Drug Discovery in Fungi
269
conditions has been shown to allow growth of
“unculturable” organisms [e.g., iChip device
(Nichols et al. 2010)] and to mine and isolate
their BGCs.
II. Molecular Biology Methods for
Fungal Natural Product Discovery
A. Manipulation of Regulatory Elements
1. Global Regulators of Fungal Secondary
Metabolism
Secondary metabolism in filamentous fungi, as
well as in actinobacteria, is often associated
with developmental maturity and a transition
to a generative (sporulating) growth mode
(Calvo et al. 2002; Wang 2003). It is therefore
unsurprising that the master regulators that
have a profound impact on the SM in filamentous fungi act pleiotropically and often have an
effect on fungal development and/or stress
response mechanisms (Garcı ´a-Estrada et al.
2018).
It is important to note that while the term ‘global’ is
conventionally used to describe elements with a regulatory effect that is not limited to a single cluster, no
regulator has been described so far that controls all,
rather than a fraction, of SM pathways in a given
organism.
Beginning with the observation that veA, a
gene previously known for its role in the control of the light-dependent morphological
switch between sexual and asexual development in Aspergillus nidulans, has an impact
on the biosynthesis of sterigmatocystin and
penicillin (Kato et al. 2003), researchers in the
field have established a direct link between
morphology, primary metabolism, and the
regulatory response in secondary metabolism.
The velvet domain-containing family of
proteins is conserved within the fungal kingdom (Bayram and Braus 2012). The members of
this family, namely, veA and velB, interact with
a methyltransferase domain-containing nuclear
protein laeA, which was initially found to be
essential for the production of sterigmatocystin
(carcinogen), and penicillin (antibiotic) (Bok
and Keller 2004). Since the discovery of laeA,
this regulatory element has been characterized
in other fungi, where its deletion led to the loss
of production of SMs, such as gliotoxin in
Aspergillus fumigatus (Keller et al. 2006), the
antihypercholesterolemic lovastatin in Aspergillus terreus, as well as aflatoxin and aflatrem
in Aspergillus flavus (Bok et al. 2006a). The laeA
regulome investigation helped correctly identify the biosynthetic gene cluster of the antitumor compound terrequinone A, previously
undescribed in A. nidulans (Bok et al. 2006b;
Bouhired et al. 2007). Similarly, Hong and coworkers identified Aspergillus fumisynnematus
to be a producer of cyclopiazonic acid upon
overexpression of laeA (Hong et al. 2015),
underlining the potential of manipulation of
this global regulator for the purpose of discovery of novel metabolites even in strains with no
readily available genome sequence. For a further overview of research focused on the laeA
regulation of SM, the reader is directed to Yin
and Keller (2011).
Another interesting discovery was that of a
transcriptional regulator designated mcrA
(multicluster regulator) in Aspergillus nidulans
(Oakley et al. 2017). The authors used a mutagenic approach to find genes that trans-activated the backbone enzymes of biosynthetic
gene clusters, which led to the discovery of a
transcription factor with a global repressive
effect on secondary metabolism. When deleted,
the mutant strain not only shows a different
profile of known SMs from the parental strain
but also produces two new cichorine derivatives, along with trace amounts of felinone A,
an antibiotic compound recently discovered in
Beauveria felina (Du et al. 2014). It has also
been shown that mcrA works independently of
laeA, as its deletion stimulates SM biosynthesis
regardless of the presence of laeA; furthermore,
new masses were observed when laeA was overexpressed in a mcrA deletion background.
Expectedly, the deletion of mcrA orthologues
in other fungal strains, namely, A. terreus and
Penicillium canescens, also led to altered metabolic profiles in the nascent mutants (Oakley
et al. 2017). Given the high level of conservation
of mcrA among ascomycetes, it is likely that the
11 New Avenues Toward Drug Discovery in Fungi
269