BGCs are silent; secondly, many producer species are difficult to propagate in bioreactors;
thirdly, many compounds are produced in
tiny amounts; fourthly, no genetic toolbox
exists for new natural production hosts; and
finally, the “generally recognized as safe”
(GRAS) production status may be more difficult to achieve with a new species.
Heterologous expression of fungal BCGs
using synthetic biology based approaches in
well-characterized fungal cell factories provides
avenues to speed up SM discovery, characterization, and production. On the other hand,
heterologous SM production is challenged by
several features including toxicity of some SMs,
poor gene annotations, intron splicing differences, compartmentalization of pathways, and
the requirement for simultaneous expression
of many SM genes. Unlike heterologous production of industrial enzymes, heterologous
production of SMs is still in its infancy, and
most studies are aiming at product discovery
and pathway elucidation rather than large-scale
production. In this section, we will present
examples on how heterologous SM-gene
expression has contributed to expand our
insights into SM biosynthesis, as well as challenges toward their production.
A. Challenges in Heterologous Secondary
Metabolite Production
1. Product Toxicity
Many SMs are antimicrobials, and they may
therefore impair the growth of, or even kill,
the new heterologous producer strain. In case
the task is to produce large amounts of a known
SM, the first experiment should therefore be to
test whether the host can tolerate the desired
SM. If SM toxicity is a problem, it may be
necessary to develop a resistant strain. If the
resistance mechanism is known in the native
producer species, it may be possible to transfer
the resistance mechanism from the native host
to the new producer strain.
For example, mycophenolic acid (MPA) from Penicillium brevicompactum kills A. nidulans by inhibiting its
inosine-5
0 -monophosphate dehydrogenase (IMPDH).
However, A. nidulans can be engineered to tolerate
mycophenolic acid by inserting mpaF of the P. brevicompactum mpa BCG, which encodes an MPAinsensitive IMPDH, into its genome (Hansen et al.
2011a).
Toxicity can also be avoided or reduced by
introducing a pump that exports the new compound. For example, the gliotoxin sensitivity of
a sirodesmin transporter-deficient strain of
Leptosphaeria maculans can be rescued by
introduction of the transporter gene gliA from
A. fumigatus (Gardiner et al. 2005). Similarly, S.
cerevisiae expressing the mlcE efflux pump
gene from the compactin BGC is protected
against statins (Ley et al. 2015). A different
mode of detoxification is based on glycosylation of the SM, a principle which is commonly
used in plants (Sandermann Jr. 1992), but
which has also been observed in fungi. For
example, during yanuthone production by A.
niger, a toxic intermediate is glycosylated
(Holm et al. 2014), or during co-culturing, Trichoderma species uses glycosylation as a
defense against deoxynivalenol, a toxin produced by Fusarium graminearum (Tian et al.
2016). For cell factory construction, this strategy has been applied for vanillin production in
S. cerevisiae by expressing a gene encoding a
glycosyltransferase from Arabidopsis thaliana
(Brochado et al. 2010).
If the task of heterologous production is
product discovery, rather than large-scale production, toxicity issues may be less important
as only small amounts are sufficient for product
identification. However, if weak growth of the
new cell factory is observed, it may be advantageous to use inducible promoters to control
expression of key genes in the pathway. For
example, the inducible alcA promoter was
fused to genes in the asperfuranone BGC to
avoid toxicity issues during pathway elucidation (Chiang et al. 2013).
250
J. K. H. Rendsvig et al.
thirdly, many compounds are produced in
tiny amounts; fourthly, no genetic toolbox
exists for new natural production hosts; and
finally, the “generally recognized as safe”
(GRAS) production status may be more difficult to achieve with a new species.
Heterologous expression of fungal BCGs
using synthetic biology based approaches in
well-characterized fungal cell factories provides
avenues to speed up SM discovery, characterization, and production. On the other hand,
heterologous SM production is challenged by
several features including toxicity of some SMs,
poor gene annotations, intron splicing differences, compartmentalization of pathways, and
the requirement for simultaneous expression
of many SM genes. Unlike heterologous production of industrial enzymes, heterologous
production of SMs is still in its infancy, and
most studies are aiming at product discovery
and pathway elucidation rather than large-scale
production. In this section, we will present
examples on how heterologous SM-gene
expression has contributed to expand our
insights into SM biosynthesis, as well as challenges toward their production.
A. Challenges in Heterologous Secondary
Metabolite Production
1. Product Toxicity
Many SMs are antimicrobials, and they may
therefore impair the growth of, or even kill,
the new heterologous producer strain. In case
the task is to produce large amounts of a known
SM, the first experiment should therefore be to
test whether the host can tolerate the desired
SM. If SM toxicity is a problem, it may be
necessary to develop a resistant strain. If the
resistance mechanism is known in the native
producer species, it may be possible to transfer
the resistance mechanism from the native host
to the new producer strain.
For example, mycophenolic acid (MPA) from Penicillium brevicompactum kills A. nidulans by inhibiting its
inosine-5
0 -monophosphate dehydrogenase (IMPDH).
However, A. nidulans can be engineered to tolerate
mycophenolic acid by inserting mpaF of the P. brevicompactum mpa BCG, which encodes an MPAinsensitive IMPDH, into its genome (Hansen et al.
2011a).
Toxicity can also be avoided or reduced by
introducing a pump that exports the new compound. For example, the gliotoxin sensitivity of
a sirodesmin transporter-deficient strain of
Leptosphaeria maculans can be rescued by
introduction of the transporter gene gliA from
A. fumigatus (Gardiner et al. 2005). Similarly, S.
cerevisiae expressing the mlcE efflux pump
gene from the compactin BGC is protected
against statins (Ley et al. 2015). A different
mode of detoxification is based on glycosylation of the SM, a principle which is commonly
used in plants (Sandermann Jr. 1992), but
which has also been observed in fungi. For
example, during yanuthone production by A.
niger, a toxic intermediate is glycosylated
(Holm et al. 2014), or during co-culturing, Trichoderma species uses glycosylation as a
defense against deoxynivalenol, a toxin produced by Fusarium graminearum (Tian et al.
2016). For cell factory construction, this strategy has been applied for vanillin production in
S. cerevisiae by expressing a gene encoding a
glycosyltransferase from Arabidopsis thaliana
(Brochado et al. 2010).
If the task of heterologous production is
product discovery, rather than large-scale production, toxicity issues may be less important
as only small amounts are sufficient for product
identification. However, if weak growth of the
new cell factory is observed, it may be advantageous to use inducible promoters to control
expression of key genes in the pathway. For
example, the inducible alcA promoter was
fused to genes in the asperfuranone BGC to
avoid toxicity issues during pathway elucidation (Chiang et al. 2013).
250
J. K. H. Rendsvig et al.