C. Molecular Engineering of Biosynthetic
Clusters and Combinatorial Synthetic
Biology
1. Manipulation of Promoter Systems
The vast economic relevance of fungi in the
biotech industry, as well as complex basic and
applied research endeavors over several decades have prompted a need for tunable gene
expression systems. The ever-improving mechanistic understanding of gene regulation has
enabled the development of promoter structures that facilitate the initiation of transcription of a gene of interest in a constitutive or
inducible manner.
Features typical for inducible promoters
include tight and controllable regulation, costoptimal induction possibility, and a high
expression of the gene downstream of the promoter (Kluge et al. 2018). Such systems developed over years have mostly been dependent on
the metabolic activity of the induced organism
and the carbon source present in the medium,
e.g., the xylP promoter induced by xylose and
repressed by glucose (Zadra et al. 2000) or the
alcA promoter induced by ethanol and
repressed by glucose (Waring et al. 1989). The
latter was used in a pioneering attempt to activate a silent SM cluster in A. nidulans by putting the cognate cluster regulator, apdR under
the control of the alcA promoter. This strategy
led to the discovery of two novel compounds,
aspyridones A and B (Bergmann et al. 2007).
Recently, the cbhI promoter, induced by various plant
saccharides, was cloned to control the heterologous
expression of two feruloyl esterase genes in Trichoderma reesei in order to liberate ferulate from wheat
bran.
Inducible promoters that do not depend on
the carbon source circumvent the obvious
obstacles of manipulating growth conditions.
Among these, the tetracycline-inducible promoter, originally based on the bacterial tetracycline resistance operon, was optimized for
eukaryotes and has been in use for decades
now (reviewed by Kluge et al. 2018). Tet-On
and Tet-Off (active in presence and absence of
a tetracycline core structure, respectively) promoters have been optimized for a number of
filamentous fungi (Vogt et al. 2005; Meyer et al.
2011; Wanka et al. 2016; Janevska et al. 2017),
ensuring the possibility of a fine-tuned expression regulation. Sun et al. (2016) conducted an
elegant study in which the Tet-On promoter
was used to overexpress the last uncharacterized PKS-NRPS hybrid gene in A. terreus. This
approach, further validated by heterologous
expression in A. nidulans, suggests that pgnA
is the only gene necessary for the production of
the metabolite.
Other particular examples of the use of the tetracyclineinducible promoter system are discussed throughout
the present work.
Recently, a novel nitrate-inducible promoter system tailored for secondary metabolite
expression, designated “CoIN” was developed
in A. nidulans (Wiemann et al. 2018). Utilizing
the bidirectional niiA/niaD promoter and supplementing the growth medium with nitrate,
the authors were capable of expressing the bcarotene pathway from Fusarium fujikuroi in
A. nidulans.
2. Combinatorial Hybrid Biosynthetic Cluster
Engineering
A particularly interesting attitude to the discovery of novel fungal natural products is the rational genetic engineering of novel enzymes
comprising the domains of biosynthetic genes
from distinct clusters. These chimeric enzymes
are then transferred into a tractable heterologous host for expression. Inspired by the
combinatorial biosynthesis approaches long
used in the research of actinobacteria (Weissman 2016), domain exchanges and module
swapping have helped create novel “unnatural”
products since as early as 1985, when mederrhodins, hybrid antibiotics from Streptomyces
sp. were reported (Omura et al. 1986).
Another hybrid PKS product of mutational biosynthesis is doramectin, an antiparasitic molecule widely used
in veterinary medicine today (Goudie et al. 1993).
11 New Avenues Toward Drug Discovery in Fungi
273
Clusters and Combinatorial Synthetic
Biology
1. Manipulation of Promoter Systems
The vast economic relevance of fungi in the
biotech industry, as well as complex basic and
applied research endeavors over several decades have prompted a need for tunable gene
expression systems. The ever-improving mechanistic understanding of gene regulation has
enabled the development of promoter structures that facilitate the initiation of transcription of a gene of interest in a constitutive or
inducible manner.
Features typical for inducible promoters
include tight and controllable regulation, costoptimal induction possibility, and a high
expression of the gene downstream of the promoter (Kluge et al. 2018). Such systems developed over years have mostly been dependent on
the metabolic activity of the induced organism
and the carbon source present in the medium,
e.g., the xylP promoter induced by xylose and
repressed by glucose (Zadra et al. 2000) or the
alcA promoter induced by ethanol and
repressed by glucose (Waring et al. 1989). The
latter was used in a pioneering attempt to activate a silent SM cluster in A. nidulans by putting the cognate cluster regulator, apdR under
the control of the alcA promoter. This strategy
led to the discovery of two novel compounds,
aspyridones A and B (Bergmann et al. 2007).
Recently, the cbhI promoter, induced by various plant
saccharides, was cloned to control the heterologous
expression of two feruloyl esterase genes in Trichoderma reesei in order to liberate ferulate from wheat
bran.
Inducible promoters that do not depend on
the carbon source circumvent the obvious
obstacles of manipulating growth conditions.
Among these, the tetracycline-inducible promoter, originally based on the bacterial tetracycline resistance operon, was optimized for
eukaryotes and has been in use for decades
now (reviewed by Kluge et al. 2018). Tet-On
and Tet-Off (active in presence and absence of
a tetracycline core structure, respectively) promoters have been optimized for a number of
filamentous fungi (Vogt et al. 2005; Meyer et al.
2011; Wanka et al. 2016; Janevska et al. 2017),
ensuring the possibility of a fine-tuned expression regulation. Sun et al. (2016) conducted an
elegant study in which the Tet-On promoter
was used to overexpress the last uncharacterized PKS-NRPS hybrid gene in A. terreus. This
approach, further validated by heterologous
expression in A. nidulans, suggests that pgnA
is the only gene necessary for the production of
the metabolite.
Other particular examples of the use of the tetracyclineinducible promoter system are discussed throughout
the present work.
Recently, a novel nitrate-inducible promoter system tailored for secondary metabolite
expression, designated “CoIN” was developed
in A. nidulans (Wiemann et al. 2018). Utilizing
the bidirectional niiA/niaD promoter and supplementing the growth medium with nitrate,
the authors were capable of expressing the bcarotene pathway from Fusarium fujikuroi in
A. nidulans.
2. Combinatorial Hybrid Biosynthetic Cluster
Engineering
A particularly interesting attitude to the discovery of novel fungal natural products is the rational genetic engineering of novel enzymes
comprising the domains of biosynthetic genes
from distinct clusters. These chimeric enzymes
are then transferred into a tractable heterologous host for expression. Inspired by the
combinatorial biosynthesis approaches long
used in the research of actinobacteria (Weissman 2016), domain exchanges and module
swapping have helped create novel “unnatural”
products since as early as 1985, when mederrhodins, hybrid antibiotics from Streptomyces
sp. were reported (Omura et al. 1986).
Another hybrid PKS product of mutational biosynthesis is doramectin, an antiparasitic molecule widely used
in veterinary medicine today (Goudie et al. 1993).
11 New Avenues Toward Drug Discovery in Fungi
273