The PKS and NRPS biosynthetic genes in
fungi share a similar modularity to those in
bacteria, facilitating their genetic shuffling and
combination as building blocks into novel protein chimeras with desired catalytic properties
(Nielsen et al. 2016). With the advancing structural information on fungal SM genes, it has
become possible to predict the enzyme programming and product synthesis (Hertweck
2015).
An inviting type of biosynthetic enzymes
presents itself in the fungal PKS-NRPS hybrids:
multimodular enzymes, usually consisting of a
type I iterative highly reducing PKS and a single
NRPS module. These naturally hybrid enzymes
are known to produce highly bioactive compounds, including cyclopiazonic acid (Seidler
et al. 1989), pseurotin (Wiemann et al. 2013a),
or fusarin C (Sondergaard et al. 2011). Given
their inherently dual character, researchers
have successfully attempted to combine the
PKS and NRPS modules from different species,
or even the same organism. Bassianin, an
extinct metabolite (i.e., a previously reported
metabolite whose producing strain is unavailable) from Beauveria bassiana, was brought
back by domain and module swapping between
two genes from the fungus, tenS and dmbS,
responsible for tenellin and desmethylbassianin
production, respectively (Heneghan et al. 2010;
Fisch et al. 2011).
A hybrid compound incorporating a tryptophan residue into the aspyridone polyketide backbone was generated by an ApdA (aspyridone synthase) and CpaS
(cyclopiazonate synthetase) chimeric enzyme (Xu
et al. 2010).
A more comprehensive approach was
employed, where PKS-NRPS compatibility was
studied by constructing 34 distinct module
swaps between five genes from four different
organisms. Many expected chimeric compounds were detected and the approach yielded
six novel metabolites (Kakule et al. 2014). A
rather interesting set of results was provided
by Nielsen et al. (2016), who used the cytochalasin PKS-NRPS hybrid, ccsA, along with a
trans-acting enoyl reductase ccsC, to create chimeras by module swapping with syn2, an
uncharacterized PKS-NRPS from Magnaporthe
oryzae, leading to identification of novel products in A. nidulans as the heterologous host.
By reciprocally combining the PKS and NRPS modules
of the respective genes, they created heterologous host
mutants expressing functional fusion proteins producing niduchimaeralin A (chimeric ccsA-syn2 PKSNRPS), and a structurally similar niduchimaeralin B
(chimeric syn2-ccsA PKS-NRPS). The authors speculate
that the success of this study depended on the structural similarity of the intermediates, rather than protein
compatibility, based on the ability of non-native condensation domain to recognize the polyketide.
This strategy to generate novel fungal chemistries is therefore potentially valuable but
inherently limited by the structural affinities
and substrate preferences of the nascent hybrid
biosynthetic enzymes.
Engineering fungal SM enzymes is by no
means limited to PKS-NRPS hybrid pathways.
Previously, bacterial NRPSs have been manipulated to generate functional recombinant
enzymes (e.g., Owen et al. 2016; Fischbach
et al. 2007), while their fungal counterparts
remained understudied. Three NRPS-like
genes from A. terreus responsible for the production of aspulvinone E, butyrolactone IIa,
and phenguignardic acid were engineered by
Wang and coworkers, swapping individual adenylation (A), thiolation (T), and thioesterase
(TE) domains (van Dijk et al. 2016). This
approach resulted in the finding that NRPSlike genes can be engineered to yield functional
hybrid enzymes that synthesize a novel metabolite.
Specifically, exchanging the A and T domain in butyrolactone IIa synthetase for phenguignardate A or A-T
domain fragment results in the production of a nonhydroxylated metabolite phenylbutyrolactone, suggesting that the function of the TE domain is independent
of the A domain.
D. Novel Techniques for Natural Product
Discovery
1. CRISPR-Cas9 in Filamentous Fungi
The innovation in the field of molecular biology
has overcome the restrictions imposed by the
274
M. Flak et al.
fungi share a similar modularity to those in
bacteria, facilitating their genetic shuffling and
combination as building blocks into novel protein chimeras with desired catalytic properties
(Nielsen et al. 2016). With the advancing structural information on fungal SM genes, it has
become possible to predict the enzyme programming and product synthesis (Hertweck
2015).
An inviting type of biosynthetic enzymes
presents itself in the fungal PKS-NRPS hybrids:
multimodular enzymes, usually consisting of a
type I iterative highly reducing PKS and a single
NRPS module. These naturally hybrid enzymes
are known to produce highly bioactive compounds, including cyclopiazonic acid (Seidler
et al. 1989), pseurotin (Wiemann et al. 2013a),
or fusarin C (Sondergaard et al. 2011). Given
their inherently dual character, researchers
have successfully attempted to combine the
PKS and NRPS modules from different species,
or even the same organism. Bassianin, an
extinct metabolite (i.e., a previously reported
metabolite whose producing strain is unavailable) from Beauveria bassiana, was brought
back by domain and module swapping between
two genes from the fungus, tenS and dmbS,
responsible for tenellin and desmethylbassianin
production, respectively (Heneghan et al. 2010;
Fisch et al. 2011).
A hybrid compound incorporating a tryptophan residue into the aspyridone polyketide backbone was generated by an ApdA (aspyridone synthase) and CpaS
(cyclopiazonate synthetase) chimeric enzyme (Xu
et al. 2010).
A more comprehensive approach was
employed, where PKS-NRPS compatibility was
studied by constructing 34 distinct module
swaps between five genes from four different
organisms. Many expected chimeric compounds were detected and the approach yielded
six novel metabolites (Kakule et al. 2014). A
rather interesting set of results was provided
by Nielsen et al. (2016), who used the cytochalasin PKS-NRPS hybrid, ccsA, along with a
trans-acting enoyl reductase ccsC, to create chimeras by module swapping with syn2, an
uncharacterized PKS-NRPS from Magnaporthe
oryzae, leading to identification of novel products in A. nidulans as the heterologous host.
By reciprocally combining the PKS and NRPS modules
of the respective genes, they created heterologous host
mutants expressing functional fusion proteins producing niduchimaeralin A (chimeric ccsA-syn2 PKSNRPS), and a structurally similar niduchimaeralin B
(chimeric syn2-ccsA PKS-NRPS). The authors speculate
that the success of this study depended on the structural similarity of the intermediates, rather than protein
compatibility, based on the ability of non-native condensation domain to recognize the polyketide.
This strategy to generate novel fungal chemistries is therefore potentially valuable but
inherently limited by the structural affinities
and substrate preferences of the nascent hybrid
biosynthetic enzymes.
Engineering fungal SM enzymes is by no
means limited to PKS-NRPS hybrid pathways.
Previously, bacterial NRPSs have been manipulated to generate functional recombinant
enzymes (e.g., Owen et al. 2016; Fischbach
et al. 2007), while their fungal counterparts
remained understudied. Three NRPS-like
genes from A. terreus responsible for the production of aspulvinone E, butyrolactone IIa,
and phenguignardic acid were engineered by
Wang and coworkers, swapping individual adenylation (A), thiolation (T), and thioesterase
(TE) domains (van Dijk et al. 2016). This
approach resulted in the finding that NRPSlike genes can be engineered to yield functional
hybrid enzymes that synthesize a novel metabolite.
Specifically, exchanging the A and T domain in butyrolactone IIa synthetase for phenguignardate A or A-T
domain fragment results in the production of a nonhydroxylated metabolite phenylbutyrolactone, suggesting that the function of the TE domain is independent
of the A domain.
D. Novel Techniques for Natural Product
Discovery
1. CRISPR-Cas9 in Filamentous Fungi
The innovation in the field of molecular biology
has overcome the restrictions imposed by the
274
M. Flak et al.