are assumed to be responsible for the high
native production of equisetin in Fusarium heterosporum. As strong transcriptional activators,
these elements were exploited to resurrect an
“extinct” pathway (i.e., a pathway from a producing organism that is no longer available)
responsible for the production of pyrrolocin
and its two derivatives, all with an antituberculosis activity (Kakule et al. 2015). In a similar
approach, the A. terreus terrein cluster transcription factor terR combined with the transcriptional control of the native promoters,
served as a basis for the development of a
strong constitutive heterologous expression
system (Gressler et al. 2015).
Further example of genetic manipulation of
regulatory genes involves the overexpression of
two pathways in A. fumigatus. (Ko ¨nig et al.
2013) generated a strain overproducing fumicyclines, originally a product induced in a bacterial co-cultivation, by putting the cluster’s
associated transcription factor under the control of the inducible icl promoter. Similarly,
fumipyrrole was found as the product of a protein kinase A/cAMP-signaling pathwayactivated NRPS cluster (Macheleidt et al.
2015). The Tet-On promoter-facilitated overexpression of the cluster-specific transcription
factor activated the biosynthesis of fumipyrrole
even in absence of an active cAMP signaling
cascade.
A. nidulans, similarly, produced asperfuranones as a
result of replacement of the native promoter of one of
the cluster’s own transcription factors by the alcohol
dehydrogenase promoter (Chiang et al. 2009).
Relying on the activating properties of
many transcription factors in fungi, other studies opted for constitutive, rather than inducible,
expression of transcription factors in order to
awaken the entire cluster. A novel diterpene
gene cluster was characterized after gpdAinduced expression of a newly discovered transcription factor pbcR in A. nidulans. While its
overexpression generally attenuated secondary
metabolite production, the seven genes adjacent to the cluster were shown to produce entpimara-8,14(15)-diene (Bromann et al. 2012).
Similarly, when the gpdA promoter was exchanged for
the native promoter of berA, a transcription factor of a
paraherquonin-like cluster encoded in Neosartorya glabra (Zhang et al. 2018), the pathway activation led to
the discovery of four berkeleyacetal derivatives.
It needs to be noted that while the selected
published examples suggest that the induction
of regulatory proteins is sufficient for the activation of a silent secondary metabolite cluster,
this is not always the case. Ahuja et al. (2012)
systematically replaced the promoters of transcription factors of 18 non-reducing PKS gene
clusters in A. nidulans with an inducible alcA
promoter. Surprisingly, this approach only
worked in three instances and yielded no or
very low increases in the production of the
remaining 15 respective SMs. Thus, additional
considerations and genetic engineering methods are necessary and have indeed proved vital
in the process of activating silent gene clusters
in fungi. This necessity is elegantly illustrated
by Grau et al. (2018). In their work, they
exchanged the DNA-binding domain of a
highly active TF associated with the asperfuranone gene cluster for the DNA-binding domain
of a silent cluster-specific TF. This hybrid synthetic regulator was able to activate the aln
cluster, producing the anti-inflammatory, antitumor polyketide asperlin, as well as a photoprotective carboxylic acid intermediate,
potentially useful in cosmetics.
B. Heterologous Expression of Fungal
Biosynthetic Gene Clusters
Expression of both, bacterial and fungal natural
product biosynthetic pathways in heterologous
hosts, has been established as a powerful technique for discovery, as well as production of
secondary metabolites (Alberti et al. 2017).
With the explosion of genomic data availability
and remarkable advances in synthetic biology
tools, heterologous expression has become a
viable alternative to total synthesis in many
cases. Numerous advantages of this approach,
including state-of-the-art molecular tools for
SM production enhancement, as well as the
11 New Avenues Toward Drug Discovery in Fungi
271
native production of equisetin in Fusarium heterosporum. As strong transcriptional activators,
these elements were exploited to resurrect an
“extinct” pathway (i.e., a pathway from a producing organism that is no longer available)
responsible for the production of pyrrolocin
and its two derivatives, all with an antituberculosis activity (Kakule et al. 2015). In a similar
approach, the A. terreus terrein cluster transcription factor terR combined with the transcriptional control of the native promoters,
served as a basis for the development of a
strong constitutive heterologous expression
system (Gressler et al. 2015).
Further example of genetic manipulation of
regulatory genes involves the overexpression of
two pathways in A. fumigatus. (Ko ¨nig et al.
2013) generated a strain overproducing fumicyclines, originally a product induced in a bacterial co-cultivation, by putting the cluster’s
associated transcription factor under the control of the inducible icl promoter. Similarly,
fumipyrrole was found as the product of a protein kinase A/cAMP-signaling pathwayactivated NRPS cluster (Macheleidt et al.
2015). The Tet-On promoter-facilitated overexpression of the cluster-specific transcription
factor activated the biosynthesis of fumipyrrole
even in absence of an active cAMP signaling
cascade.
A. nidulans, similarly, produced asperfuranones as a
result of replacement of the native promoter of one of
the cluster’s own transcription factors by the alcohol
dehydrogenase promoter (Chiang et al. 2009).
Relying on the activating properties of
many transcription factors in fungi, other studies opted for constitutive, rather than inducible,
expression of transcription factors in order to
awaken the entire cluster. A novel diterpene
gene cluster was characterized after gpdAinduced expression of a newly discovered transcription factor pbcR in A. nidulans. While its
overexpression generally attenuated secondary
metabolite production, the seven genes adjacent to the cluster were shown to produce entpimara-8,14(15)-diene (Bromann et al. 2012).
Similarly, when the gpdA promoter was exchanged for
the native promoter of berA, a transcription factor of a
paraherquonin-like cluster encoded in Neosartorya glabra (Zhang et al. 2018), the pathway activation led to
the discovery of four berkeleyacetal derivatives.
It needs to be noted that while the selected
published examples suggest that the induction
of regulatory proteins is sufficient for the activation of a silent secondary metabolite cluster,
this is not always the case. Ahuja et al. (2012)
systematically replaced the promoters of transcription factors of 18 non-reducing PKS gene
clusters in A. nidulans with an inducible alcA
promoter. Surprisingly, this approach only
worked in three instances and yielded no or
very low increases in the production of the
remaining 15 respective SMs. Thus, additional
considerations and genetic engineering methods are necessary and have indeed proved vital
in the process of activating silent gene clusters
in fungi. This necessity is elegantly illustrated
by Grau et al. (2018). In their work, they
exchanged the DNA-binding domain of a
highly active TF associated with the asperfuranone gene cluster for the DNA-binding domain
of a silent cluster-specific TF. This hybrid synthetic regulator was able to activate the aln
cluster, producing the anti-inflammatory, antitumor polyketide asperlin, as well as a photoprotective carboxylic acid intermediate,
potentially useful in cosmetics.
B. Heterologous Expression of Fungal
Biosynthetic Gene Clusters
Expression of both, bacterial and fungal natural
product biosynthetic pathways in heterologous
hosts, has been established as a powerful technique for discovery, as well as production of
secondary metabolites (Alberti et al. 2017).
With the explosion of genomic data availability
and remarkable advances in synthetic biology
tools, heterologous expression has become a
viable alternative to total synthesis in many
cases. Numerous advantages of this approach,
including state-of-the-art molecular tools for
SM production enhancement, as well as the
11 New Avenues Toward Drug Discovery in Fungi
271