potential to refactor the natural pathways to
synthesize novel product analogs, render this
practice attractive in the field of fungal research
(Billingsley et al. 2016). Heterologous expression is also considered to be the optimal tactic
in the attempts to elucidate biosynthetic pathways in organisms exhibiting slow growth
under laboratory conditions, which are not yet
culturable, or in those that are not genetically
amenable at present (Schmidt-Dannert 2015).
Multiple criteria must be considered and
addressed in order to achieve a successful
expression of a given pathway in a non-native
host. Careful appraisal of potentially problematic sequence elements such as introns, along
with codon optimization and the choice of a
suitable host, may prove crucial (Billingsley
et al. 2016). Generally, selecting a related organism naturally possessing the ability to produce
secondary metabolites improves the chances of
success in expression of an exogenous pathway
(Mattern et al. 2015b). It is, however, important
to acknowledge that even microorganisms
devoid of secondary metabolism can be engineered for production of a wide array of bioactive secondary metabolites. This was the case
with Saccharomyces cerevisiae, a biotechnology
workhorse, producing the fungal polyketide
lovastatin (Ley et al. 2015), or even the plantderived antimalarial terpenoid artemisinin
(Arsenault et al. 2008) or alkaloid hydrocodone
(Galanie et al. 2015).
A recent work by Hillenmeyer and coworkers established a large-scale heterologous SM
expression platform (HEx) in yeast (Harvey
et al. 2018). The authors scanned all available
fungal genomes to date to identify 3512 biosynthetic gene clusters from which 41 previously
uncharacterized clusters were selected (from
both ascomycetes and basidiomycetes), harboring unique PKS and UbiA-like terpene cyclase
(UTC) core genes. The constituent genes from
these clusters, placed under the control of the
yeast P ADH2 promoter, were cloned into highly
optimized S. cerevisiae strains using plasmid
vectors. Twenty-two heterologous expression
strains were reported to synthesize compounds
absent in the control strains, 11 of these
appeared to be completely novel. The structure
of these new compounds was elucidated; however, none were tested for bioactive properties.
Nevertheless, this effort underlines the power of
heterologous expression of secondary metabolite gene clusters.
Among filamentous fungi, Aspergilli have
proved to be particularly rewarding hosts for
heterologous expression of biosynthetic gene
clusters. Aspergillus oryzae has been used to
heterologously synthesize high yields of antibacterial citrinin from Monascus purpureus (Sakai
et al. 2008), an inhibitor of potassium pump
with antibacterial effect paxilline from Penicillium paxilli (Tagami et al. 2014), or pleuromutilin, an antimicrobial compound from
Clitopilus passeckerianus (Bailey et al. 2016).
A similar approach has been used for the discovery of a
multitude of novel compounds, examples ranging from
prenylated cyclic dipeptides produced in A. nidulans
(Wunsch et al. 2015), to two novel ergot alkaloids being
produced in A. fumigatus (Robinson and Panaccione
2014). Other examples of heterologous expression in
filamentous fungi have been extensively reviewed in the
past (Schu ¨mann and Hertweck 2006; Lubertozzi and
Keasling 2009; Anyaogu and Mortensen 2015).
Of special note is the report of a modification of the product of known pathways by
the heterologous host, raising the question
whether compounds discovered using this
approach with other, uncharacterized clusters,
truly represent their bona fide products. The
expression of the cytochalasin synthetase of A.
clavatus, surprisingly, led to the production of a
modified intermediate, niduclavine, in A. nidulans (Nielsen et al. 2016).
From a prognostic point of view, it is reasonable to assume that filamentous fungi (as
well as their relatives from other clades) will
play a consequential role in basic and applied
research, as well as industrial production of
pharmaceuticals and agrochemicals, be it in
the role of heterologous expression hosts,
ample sources for new compounds discovery,
or as platforms for directed synthetic biology
aimed at novel compound analogs with desired
properties.
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M. Flak et al.
synthesize novel product analogs, render this
practice attractive in the field of fungal research
(Billingsley et al. 2016). Heterologous expression is also considered to be the optimal tactic
in the attempts to elucidate biosynthetic pathways in organisms exhibiting slow growth
under laboratory conditions, which are not yet
culturable, or in those that are not genetically
amenable at present (Schmidt-Dannert 2015).
Multiple criteria must be considered and
addressed in order to achieve a successful
expression of a given pathway in a non-native
host. Careful appraisal of potentially problematic sequence elements such as introns, along
with codon optimization and the choice of a
suitable host, may prove crucial (Billingsley
et al. 2016). Generally, selecting a related organism naturally possessing the ability to produce
secondary metabolites improves the chances of
success in expression of an exogenous pathway
(Mattern et al. 2015b). It is, however, important
to acknowledge that even microorganisms
devoid of secondary metabolism can be engineered for production of a wide array of bioactive secondary metabolites. This was the case
with Saccharomyces cerevisiae, a biotechnology
workhorse, producing the fungal polyketide
lovastatin (Ley et al. 2015), or even the plantderived antimalarial terpenoid artemisinin
(Arsenault et al. 2008) or alkaloid hydrocodone
(Galanie et al. 2015).
A recent work by Hillenmeyer and coworkers established a large-scale heterologous SM
expression platform (HEx) in yeast (Harvey
et al. 2018). The authors scanned all available
fungal genomes to date to identify 3512 biosynthetic gene clusters from which 41 previously
uncharacterized clusters were selected (from
both ascomycetes and basidiomycetes), harboring unique PKS and UbiA-like terpene cyclase
(UTC) core genes. The constituent genes from
these clusters, placed under the control of the
yeast P ADH2 promoter, were cloned into highly
optimized S. cerevisiae strains using plasmid
vectors. Twenty-two heterologous expression
strains were reported to synthesize compounds
absent in the control strains, 11 of these
appeared to be completely novel. The structure
of these new compounds was elucidated; however, none were tested for bioactive properties.
Nevertheless, this effort underlines the power of
heterologous expression of secondary metabolite gene clusters.
Among filamentous fungi, Aspergilli have
proved to be particularly rewarding hosts for
heterologous expression of biosynthetic gene
clusters. Aspergillus oryzae has been used to
heterologously synthesize high yields of antibacterial citrinin from Monascus purpureus (Sakai
et al. 2008), an inhibitor of potassium pump
with antibacterial effect paxilline from Penicillium paxilli (Tagami et al. 2014), or pleuromutilin, an antimicrobial compound from
Clitopilus passeckerianus (Bailey et al. 2016).
A similar approach has been used for the discovery of a
multitude of novel compounds, examples ranging from
prenylated cyclic dipeptides produced in A. nidulans
(Wunsch et al. 2015), to two novel ergot alkaloids being
produced in A. fumigatus (Robinson and Panaccione
2014). Other examples of heterologous expression in
filamentous fungi have been extensively reviewed in the
past (Schu ¨mann and Hertweck 2006; Lubertozzi and
Keasling 2009; Anyaogu and Mortensen 2015).
Of special note is the report of a modification of the product of known pathways by
the heterologous host, raising the question
whether compounds discovered using this
approach with other, uncharacterized clusters,
truly represent their bona fide products. The
expression of the cytochalasin synthetase of A.
clavatus, surprisingly, led to the production of a
modified intermediate, niduclavine, in A. nidulans (Nielsen et al. 2016).
From a prognostic point of view, it is reasonable to assume that filamentous fungi (as
well as their relatives from other clades) will
play a consequential role in basic and applied
research, as well as industrial production of
pharmaceuticals and agrochemicals, be it in
the role of heterologous expression hosts,
ample sources for new compounds discovery,
or as platforms for directed synthetic biology
aimed at novel compound analogs with desired
properties.
272
M. Flak et al.