an approach combining a double promoter
exchange and polycistronic heterologous
expression to optimize the expression levels of
a reporter gene, subsequently verifying the
capacity of the system using the aspulvinone E
synthetase gene from A. terreus. The result was
a finely tunable, strong gene expression platform.
Crossing the borders of secondary metabolism
research, the 2A peptide-based system was also used
to assemble a bicistronic construct of a Penicillium
funiculosum cellobiohydrolase enzyme and a GFP,
which was introduced into T. reesei, allowing the
authors to monitor the expression of the enzyme (Subramanian et al. 2017).
Recently, Hoefgen et al. (2018) introduced
an advanced method of polycistronic expression, introducing a split fluorescent marker,
active only upon successful assembly and
allowing for simple selection of positive transformants using fluorescence microscopy, along
with a TEV protease to reduce the remaining Cterminus tag of the 2A peptides. This improved
method was used to heterologously produce
exceptionally high yields of the psychotropic
alkaloid psilocybin in A. nidulans. The hallucinogenic compound has recently regained interest, showing a pharmaceutical potential as an
anxiolytic in terminal-stage cancer patients and
antidepressant (Griffiths et al. 2016).
It is worthwhile to note that this research is
recent, and while no new fungal metabolites
have been discovered yet, it is conceivable that
the polycistronic expression of whole fungal
pathways will, in the future, yield novel secondary metabolites with interesting bioactive
potential.
3. Fungal Artificial Chromosomes
An elegant, albeit isolated technique
incorporated the idea of bacterial and yeast
artificial chromosomes and translated it to filamentous fungi, giving rise to so-called fungal
artificial chromosomes (FACs). Constructed on
a bacterial plasmid backbone harboring the
fungal autonomously replicating fragment
AMA1, these genetic elements are capable of
replicating in both E. coli and A. nidulans. Randomly sheared segments of A. terreus genomic
DNA were cloned into the backbone, creating
the FACs. All 56 annotated biosynthetic gene
clusters from A. terreus were introduced into
FACs in this fashion and successfully shuttled
into A. nidulans as a heterologous host (Bok
et al. 2015). To validate the system, the authors
analyzed a host mutant carrying the A. terreus
astechrome cluster and discovered terrezine D,
a precursor molecule in astechrome biosynthesis machinery.
The utilization of unbiased, randomly
sheared gDNA is specifically of note. This renders the FACs methodology particularly applicable in the case of unsequenced microbes and
complex microbial metagenomes and highlights its enabling potential to identify SM
gene clusters and their corresponding products.
III. Fungal Interactions as a Source of
New Compounds
A. Induction of Natural Product Formation by
Co-cultivation
At the beginning of natural product research,
isolates of fungi and other microorganisms
were taken from distinct habitats, such as soil,
grown in monoculture, and fermented to find
metabolites showing desirable activity. This
approach was highly successful and yielded an
abundance of potent pharmaceuticals, which
are still being used to date. It is widely accepted
that fungi, as well as all other microorganisms,
do not live in isolation in their natural habitat
(Fig. 11.2). Therefore, organismal interactions
are highly likely to occur and secondary metabolites are mainly thought to serve as arsenal in
the defense of an ecological niche (Netzker et al.
2018). An abundance of co-cultivation experiments has been carried out in order to obtain
novel antibiotics to refill the antibiotic pipeline,
as well as to gain insight into the biology of
organismal interactions. In the next sections
we aim to give an overview of examples in
which natural product formation was induced
276
M. Flak et al.
exchange and polycistronic heterologous
expression to optimize the expression levels of
a reporter gene, subsequently verifying the
capacity of the system using the aspulvinone E
synthetase gene from A. terreus. The result was
a finely tunable, strong gene expression platform.
Crossing the borders of secondary metabolism
research, the 2A peptide-based system was also used
to assemble a bicistronic construct of a Penicillium
funiculosum cellobiohydrolase enzyme and a GFP,
which was introduced into T. reesei, allowing the
authors to monitor the expression of the enzyme (Subramanian et al. 2017).
Recently, Hoefgen et al. (2018) introduced
an advanced method of polycistronic expression, introducing a split fluorescent marker,
active only upon successful assembly and
allowing for simple selection of positive transformants using fluorescence microscopy, along
with a TEV protease to reduce the remaining Cterminus tag of the 2A peptides. This improved
method was used to heterologously produce
exceptionally high yields of the psychotropic
alkaloid psilocybin in A. nidulans. The hallucinogenic compound has recently regained interest, showing a pharmaceutical potential as an
anxiolytic in terminal-stage cancer patients and
antidepressant (Griffiths et al. 2016).
It is worthwhile to note that this research is
recent, and while no new fungal metabolites
have been discovered yet, it is conceivable that
the polycistronic expression of whole fungal
pathways will, in the future, yield novel secondary metabolites with interesting bioactive
potential.
3. Fungal Artificial Chromosomes
An elegant, albeit isolated technique
incorporated the idea of bacterial and yeast
artificial chromosomes and translated it to filamentous fungi, giving rise to so-called fungal
artificial chromosomes (FACs). Constructed on
a bacterial plasmid backbone harboring the
fungal autonomously replicating fragment
AMA1, these genetic elements are capable of
replicating in both E. coli and A. nidulans. Randomly sheared segments of A. terreus genomic
DNA were cloned into the backbone, creating
the FACs. All 56 annotated biosynthetic gene
clusters from A. terreus were introduced into
FACs in this fashion and successfully shuttled
into A. nidulans as a heterologous host (Bok
et al. 2015). To validate the system, the authors
analyzed a host mutant carrying the A. terreus
astechrome cluster and discovered terrezine D,
a precursor molecule in astechrome biosynthesis machinery.
The utilization of unbiased, randomly
sheared gDNA is specifically of note. This renders the FACs methodology particularly applicable in the case of unsequenced microbes and
complex microbial metagenomes and highlights its enabling potential to identify SM
gene clusters and their corresponding products.
III. Fungal Interactions as a Source of
New Compounds
A. Induction of Natural Product Formation by
Co-cultivation
At the beginning of natural product research,
isolates of fungi and other microorganisms
were taken from distinct habitats, such as soil,
grown in monoculture, and fermented to find
metabolites showing desirable activity. This
approach was highly successful and yielded an
abundance of potent pharmaceuticals, which
are still being used to date. It is widely accepted
that fungi, as well as all other microorganisms,
do not live in isolation in their natural habitat
(Fig. 11.2). Therefore, organismal interactions
are highly likely to occur and secondary metabolites are mainly thought to serve as arsenal in
the defense of an ecological niche (Netzker et al.
2018). An abundance of co-cultivation experiments has been carried out in order to obtain
novel antibiotics to refill the antibiotic pipeline,
as well as to gain insight into the biology of
organismal interactions. In the next sections
we aim to give an overview of examples in
which natural product formation was induced
276
M. Flak et al.