traditional molecular toolkit used in filamentous fungi: gene deletion, overexpression, or
mutagenesis. The advent of CRISPR-Cas9 gene
editing technology (Jinek et al. 2012), based on
the bacterial adaptive immunity derived from
Streptococcus pyogenes, and suitable for multiple concurrent gene-editing events, was
promptly appropriated and installed in fungal
hosts (Krappmann 2017; Weber et al. 2017).
The system was established in the model species Neurospora crassa (Matsu-ura et al. 2015), A. nidulans
(Nødvig et al. 2015), the industrial workhorse, Trichoderma reesei (Liu et al. 2015), phytopathogenic Ustilago
maydis (Schuster et al. 2016), and the human pathogens
Candida albicans (Vyas et al. 2015) and A. fumigatus
(Fuller et al. 2015).
This technology promises to accelerate
genetic engineering by offering highly targeted,
markerless editing of DNA. Deng et al. (2017)
provides a comprehensive overview of the state
of the art of CRISPR-Cas9 technology in filamentous fungi.
In A. fumigatus in particular, the CRISPSCas9 gene editing coupled with a split marker
system has been used to reconstitute the functionality of a mutated trypacidin biosynthetic
gene cluster (Weber et al. 2017). In this study,
the authors utilized the CRISPR capability to
edit single bases within the genome and “corrected” a point mutation that resulted in a premature stop codon in the PKS-coding tynC
gene. To advance the efficiency of the method
and prevent further alterations to the locus of
interest, a spatially separated split marker was
introduced that contained a gRNA recognition
site and PAM identical to that of the tynC target
site on each fragment. Thus, the selection
marker was only active after cleavage by Cas9
endonuclease, which concurrently also introduced a double-strand break in tynC, allowing
for a recombination event with provided donor
DNA, effectively reconstituting the gene’s functionality in a formerly non-producing strain.
Trypacidin has been shown to be present in
fungal spores, and it is hypothesized to have a
protective effect against phagocytosis due to its
amoebicidal activity (Mattern et al. 2015a).
CRISPR-Cas9 technology was also used to
genetically engineer and explore the secondary
metabolism of Talaromyces atroroseus, a
fungus previously recalcitrant to genetic
manipulation. Using the CRISPR-Cas9-enabled
efficient gene targeting, the authors were able to
delete the gene encoding a hybrid PKS-NRPS,
identified as an orthologue of those producing
nitrogen-containing tetramic acid derivatives
in other filamentous fungi (Nielsen et al.
2017). This hybrid gene is responsible for the
production of talaroconvolutin A and its medically relevant stereoisomer ZG-1494a.
The latter is an inhibitor of platelet-activating factor
acetyltransferase (West et al. 1996), while talaroconvolutins have been evaluated for their antifungal activity
(Suzuki et al. 2000).
2. Polycistronic Expression of Biosynthetic
Pathways
Pioneered by the recent publication of the
introduction of a construct containing the
penicillin biosynthetic pathway genes encoded
in a single polycistronic mRNA in a nonproducing strain of A. nidulans (Unkles et al.
2014), the technique has sparked interest
among fungal molecular biologists. This novel
synthetic biology approach assembles multiple
genes in a single mRNA and utilizes viral
2A peptide sequences to facilitate a cotranslational cleavage of the nascent peptide
chain by the ribosomes. That opens the possibility of introducing entire biosynthetic gene
pathways in convenient heterologous hosts, circumventing promoter exchange limitations, the
need for individual gene overexpression, or
selection marker recycling.
This approach has since been validated and
optimized for A. niger. Enniatin production
native to Fusarium oxysporum was installed in
the biotechnology workhorse by introducing
ketoisovalerate reductase and enniatin synthetase genes, required for the SM synthesis,
linked with 2A peptide sequences (Schuetze
and Meyer 2017). Geib and Brock (2017) used
11 New Avenues Toward Drug Discovery in Fungi
275
mutagenesis. The advent of CRISPR-Cas9 gene
editing technology (Jinek et al. 2012), based on
the bacterial adaptive immunity derived from
Streptococcus pyogenes, and suitable for multiple concurrent gene-editing events, was
promptly appropriated and installed in fungal
hosts (Krappmann 2017; Weber et al. 2017).
The system was established in the model species Neurospora crassa (Matsu-ura et al. 2015), A. nidulans
(Nødvig et al. 2015), the industrial workhorse, Trichoderma reesei (Liu et al. 2015), phytopathogenic Ustilago
maydis (Schuster et al. 2016), and the human pathogens
Candida albicans (Vyas et al. 2015) and A. fumigatus
(Fuller et al. 2015).
This technology promises to accelerate
genetic engineering by offering highly targeted,
markerless editing of DNA. Deng et al. (2017)
provides a comprehensive overview of the state
of the art of CRISPR-Cas9 technology in filamentous fungi.
In A. fumigatus in particular, the CRISPSCas9 gene editing coupled with a split marker
system has been used to reconstitute the functionality of a mutated trypacidin biosynthetic
gene cluster (Weber et al. 2017). In this study,
the authors utilized the CRISPR capability to
edit single bases within the genome and “corrected” a point mutation that resulted in a premature stop codon in the PKS-coding tynC
gene. To advance the efficiency of the method
and prevent further alterations to the locus of
interest, a spatially separated split marker was
introduced that contained a gRNA recognition
site and PAM identical to that of the tynC target
site on each fragment. Thus, the selection
marker was only active after cleavage by Cas9
endonuclease, which concurrently also introduced a double-strand break in tynC, allowing
for a recombination event with provided donor
DNA, effectively reconstituting the gene’s functionality in a formerly non-producing strain.
Trypacidin has been shown to be present in
fungal spores, and it is hypothesized to have a
protective effect against phagocytosis due to its
amoebicidal activity (Mattern et al. 2015a).
CRISPR-Cas9 technology was also used to
genetically engineer and explore the secondary
metabolism of Talaromyces atroroseus, a
fungus previously recalcitrant to genetic
manipulation. Using the CRISPR-Cas9-enabled
efficient gene targeting, the authors were able to
delete the gene encoding a hybrid PKS-NRPS,
identified as an orthologue of those producing
nitrogen-containing tetramic acid derivatives
in other filamentous fungi (Nielsen et al.
2017). This hybrid gene is responsible for the
production of talaroconvolutin A and its medically relevant stereoisomer ZG-1494a.
The latter is an inhibitor of platelet-activating factor
acetyltransferase (West et al. 1996), while talaroconvolutins have been evaluated for their antifungal activity
(Suzuki et al. 2000).
2. Polycistronic Expression of Biosynthetic
Pathways
Pioneered by the recent publication of the
introduction of a construct containing the
penicillin biosynthetic pathway genes encoded
in a single polycistronic mRNA in a nonproducing strain of A. nidulans (Unkles et al.
2014), the technique has sparked interest
among fungal molecular biologists. This novel
synthetic biology approach assembles multiple
genes in a single mRNA and utilizes viral
2A peptide sequences to facilitate a cotranslational cleavage of the nascent peptide
chain by the ribosomes. That opens the possibility of introducing entire biosynthetic gene
pathways in convenient heterologous hosts, circumventing promoter exchange limitations, the
need for individual gene overexpression, or
selection marker recycling.
This approach has since been validated and
optimized for A. niger. Enniatin production
native to Fusarium oxysporum was installed in
the biotechnology workhorse by introducing
ketoisovalerate reductase and enniatin synthetase genes, required for the SM synthesis,
linked with 2A peptide sequences (Schuetze
and Meyer 2017). Geib and Brock (2017) used
11 New Avenues Toward Drug Discovery in Fungi
275