vant meroterpenoid daurichromenic acid
(DCA) from the plant Rhododendron dauricum
in A. oryzae represents a successful case of a
functional semi-synthetic pathway (Okada et al.
2017). Using fungal expression vectors for
ectopic genome integration, the production of
DCA was achieved by expressing two fungal
genes from Stachybotrys bisbyi encoding a
polyketide synthase (StbA) and a prenyltransferase (StbC), as well as a plant gene from the
native producer encoding the DCA synthase.
Similarly, production of carminic acid, an
important food colorant produced by the scale
insect Dactylopius coccus, has been achieved in
A. nidulans via a semi-synthetic pathway
(Frandsen et al. 2018). In this case, the fivestep pathway was based on a single gene from
the natural producer encoding a C-glucosyltransferase, a plant gene from Aloe arborescens
encoding a type III octaketide synthase, two
bacterial genes from Streptomyces sp. R1128
encoding a cyclase (ZhuI) and a aromatase
(ZhuJ), and finally an unknown fungal gene
from A. nidulans, which putatively encodes a
monooxygenase. Importantly, unlike the DCA
cell factory, all non-fungal genes were inserted
into fungal expression cassettes and integrated
into defined expression sites. These examples
highlight the potential of puzzling together
pathways by combining genes from different
organisms. Similar strategies may likely gain
an increasing role in future development fungal
cell factories for production of known and
novel small molecules.
VI. Concluding Remarks and
Perspectives
This review demonstrates that heterologous
production in fungi is already well established
and that synthetic biology-based methods are
increasingly used to create new cell factories,
typically via bio-block-based strategies. In the
future, we envision that this trend will gather
momentum and that libraries of bio-blocks
containing mutated genes, scrambled homologous genes, or synthetic genes encoding new
combinations of functional domains will serve
as common resources for developing cell factories that produce, e.g., industrial enzymes
with new or improved properties or SMs that
may target new diseases. In addition, increasingly efficient high-throughput CRISPR-based
methods will allow for the generation of
genome-wide mutant-strain libraries. Hence,
rather than using a single strain for construction of a heterologous cell factory, mutant
libraries may serve as the preferred starting
point toward developing new cell factories. In
combination, these strategies will create an
enormous number of potential cell factories;
and screening for the successful candidates
will very likely constitute a bottleneck. The
expanding synthetic biology toolbox may also
contribute to address this challenge. For example, it may offer biosensors that can be used to
screen for high-yielding strains. In agreement
with this view, membrane-bound RFP was
recently used as a biosensor in allowing for
high-throughput FACS-based screening of T.
reesei mutants to identify strains with improved
enzyme secretion potential (Gao et al. 2018).
Interestingly, biosensors that monitor the
levels of specific intracellular metabolites are
already available for E. coli and S. cerevisiae
(Adeniran et al. 2015; Morris 2010; Rogers
et al. 2016; Zhang and Keasling 2011), and it is
likely that this type biosensors will be used in
the future to identify superior fungal cell factories for production of specific SMs.
Novel methods for controlling gene function during fermentation are highly desirable.
To this end, we envision that new improved
orthogonal TFs with programmable specificities, e.g., by using catalytically dead Cas9 variants as TFs (Qi et al. 2013), will be developed.
Such TFs have already been shown in other
organisms to facilitate the activation of silent
genes (Cheng et al. 2013; Perez-Pinera et al.
2013), to regulate metabolism of a heterologous
cell factories for increased production yields
(Deaner and Alper 2017; Jensen et al. 2017;
Vanegas et al. 2017), to study genetic interactions (Du et al. 2017; Peters et al. 2016), or even
to induce directed evolution (Hess et al. 2016;
Ma et al. 2016). We also envision that synthetic
biology tools will change the fungal production
platforms dramatically. Specifically, highly effi254
J. K. H. Rendsvig et al.
(DCA) from the plant Rhododendron dauricum
in A. oryzae represents a successful case of a
functional semi-synthetic pathway (Okada et al.
2017). Using fungal expression vectors for
ectopic genome integration, the production of
DCA was achieved by expressing two fungal
genes from Stachybotrys bisbyi encoding a
polyketide synthase (StbA) and a prenyltransferase (StbC), as well as a plant gene from the
native producer encoding the DCA synthase.
Similarly, production of carminic acid, an
important food colorant produced by the scale
insect Dactylopius coccus, has been achieved in
A. nidulans via a semi-synthetic pathway
(Frandsen et al. 2018). In this case, the fivestep pathway was based on a single gene from
the natural producer encoding a C-glucosyltransferase, a plant gene from Aloe arborescens
encoding a type III octaketide synthase, two
bacterial genes from Streptomyces sp. R1128
encoding a cyclase (ZhuI) and a aromatase
(ZhuJ), and finally an unknown fungal gene
from A. nidulans, which putatively encodes a
monooxygenase. Importantly, unlike the DCA
cell factory, all non-fungal genes were inserted
into fungal expression cassettes and integrated
into defined expression sites. These examples
highlight the potential of puzzling together
pathways by combining genes from different
organisms. Similar strategies may likely gain
an increasing role in future development fungal
cell factories for production of known and
novel small molecules.
VI. Concluding Remarks and
Perspectives
This review demonstrates that heterologous
production in fungi is already well established
and that synthetic biology-based methods are
increasingly used to create new cell factories,
typically via bio-block-based strategies. In the
future, we envision that this trend will gather
momentum and that libraries of bio-blocks
containing mutated genes, scrambled homologous genes, or synthetic genes encoding new
combinations of functional domains will serve
as common resources for developing cell factories that produce, e.g., industrial enzymes
with new or improved properties or SMs that
may target new diseases. In addition, increasingly efficient high-throughput CRISPR-based
methods will allow for the generation of
genome-wide mutant-strain libraries. Hence,
rather than using a single strain for construction of a heterologous cell factory, mutant
libraries may serve as the preferred starting
point toward developing new cell factories. In
combination, these strategies will create an
enormous number of potential cell factories;
and screening for the successful candidates
will very likely constitute a bottleneck. The
expanding synthetic biology toolbox may also
contribute to address this challenge. For example, it may offer biosensors that can be used to
screen for high-yielding strains. In agreement
with this view, membrane-bound RFP was
recently used as a biosensor in allowing for
high-throughput FACS-based screening of T.
reesei mutants to identify strains with improved
enzyme secretion potential (Gao et al. 2018).
Interestingly, biosensors that monitor the
levels of specific intracellular metabolites are
already available for E. coli and S. cerevisiae
(Adeniran et al. 2015; Morris 2010; Rogers
et al. 2016; Zhang and Keasling 2011), and it is
likely that this type biosensors will be used in
the future to identify superior fungal cell factories for production of specific SMs.
Novel methods for controlling gene function during fermentation are highly desirable.
To this end, we envision that new improved
orthogonal TFs with programmable specificities, e.g., by using catalytically dead Cas9 variants as TFs (Qi et al. 2013), will be developed.
Such TFs have already been shown in other
organisms to facilitate the activation of silent
genes (Cheng et al. 2013; Perez-Pinera et al.
2013), to regulate metabolism of a heterologous
cell factories for increased production yields
(Deaner and Alper 2017; Jensen et al. 2017;
Vanegas et al. 2017), to study genetic interactions (Du et al. 2017; Peters et al. 2016), or even
to induce directed evolution (Hess et al. 2016;
Ma et al. 2016). We also envision that synthetic
biology tools will change the fungal production
platforms dramatically. Specifically, highly effi254
J. K. H. Rendsvig et al.