teins may also be possible in filamentous fungi
after dedicated strain engineering (Anyaogu
and Mortensen 2015).
b) Chaperones and ER Stress
Overexpression of secretory proteins may
result in accumulation of misfolded proteins
in the ER, and this problem poses a severe
bottleneck toward high yields of secreted proteins. Cells respond to misfolded proteins in the
ER by triggering the unfolded protein response
(UPR), which is sensed by the ER transmembrane protein Ire1. Normally, Ire1 forms an
inactive complex with the chaperone BipA,
but as BipA is increasingly recruited to assist
in folding, Ire1 forms a nucleolytically active
dimer that catalyzes splicing of the nonconventional intron in the hac1 mRNA (Krishnan and Askew 2014). The processed hac1
mRNA encodes mature Hac1 transcription factor, which in turn triggers transcription of UPR
genes. This includes genes encoding ERresident molecular chaperones like BipA, the
heat-shock protein family of chaperones
(Hsp104, Hsp70, Hsp90) and the protein
disulfide-isomerase PdiA, thus satisfying the
increased demand for folding capacity
(Zubieta et al. 2018). Persistently misfolded
proteins are deleterious to the cell and are
degraded through the ERAD (ER-associated
degradation) pathway that includes transport
to the cytoplasm and ubiquitin-mediated degradation by the proteasome (Carvalho et al.
2011).
Several strategies have been pursued to
optimize folding. These include defensive
methods, like lowering the growth temperature
or applying weaker promoters, but also potentially more awarding methods based on stimulating the folding potential via genetic
engineering of the host organism folding
machinery.
To promote heterologous protein production in A.
awamori, the UPR pathway has been constitutively
activated by overexpressing an intron-free variant of
the hac1 gene. Using this strain, production of Trametes versicolor laccase (Lcc1) was increased sevenfold
and bovine chymosin 2.8-fold (Valkonen et al. 2003).
Similarly, heterologous production of A. niger glucose
oxidase (Gox) in a T. reesei strain overexpressing
intron-free hac1 increased production 1.8-fold (Wu
et al. 2017). In comparison, overexpression of the
bipA chaperone alone in T. reesei increased production
of Gox by 1.5-fold.
Folding of proteins containing multiple
disulfide bonds may be particularly challenging
and may benefit from increased levels of the
protein disulfide isomerase PdiA. In agreement
with this, production of thaumatin, a sweettasting plant protein containing eight disulfide
bonds, was increased twofold by using an A.
awamori strain overexpressing pdiA (Moralejo
et al. 2001). Since heterologous protein production appears to benefit from increased levels of
host chaperones, it is tempting to speculate that
more process-specific folding assistance could
be achieved by co-expressing genes encoding
one or more chaperones from the natural
source of the protein of interest.
3. Vesicular Trafficking and Polarized Growth
Filamentous fungi propagate as hyphae and the
general view is that most protein secretion
occurs at the hyphal tip (Cairns et al. 2019).
Secreted proteins are packed into vesicles,
either by cargo-receptors or as the result of
bulk flow, thereby mediating transport from
the ER to the Golgi and from the Golgi to the
plasma membrane (Barlowe and Miller 2013).
Most vesicles accumulate at the Spitzenko ¨rper
at the apex of the hyphae before fusing to the
membrane in a process that depends on the
exocyst octamer (Ahmed et al. 2018; Riquelme
and Sa ´nchez-Leo ´n 2014). Since proteins are
synthesized throughout the hyphae, vesicles
are transported by an elaborate transport system based on actin filaments and microtubules
to ensure efficient transport to the hyphal apex
(Steinberg et al. 2017). Many of the individual
steps in the secretory pathway are understood
in molecular detail, and a popular strategy aiming at enhancing secretory transport is to overexpress genes encoding proteins that are
directly involved in transport. For example,
loading of cargo proteins into vesicles and targeting vesicles to a destination membrane have
been engineered to increase secretion as exemplified below.
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
247
after dedicated strain engineering (Anyaogu
and Mortensen 2015).
b) Chaperones and ER Stress
Overexpression of secretory proteins may
result in accumulation of misfolded proteins
in the ER, and this problem poses a severe
bottleneck toward high yields of secreted proteins. Cells respond to misfolded proteins in the
ER by triggering the unfolded protein response
(UPR), which is sensed by the ER transmembrane protein Ire1. Normally, Ire1 forms an
inactive complex with the chaperone BipA,
but as BipA is increasingly recruited to assist
in folding, Ire1 forms a nucleolytically active
dimer that catalyzes splicing of the nonconventional intron in the hac1 mRNA (Krishnan and Askew 2014). The processed hac1
mRNA encodes mature Hac1 transcription factor, which in turn triggers transcription of UPR
genes. This includes genes encoding ERresident molecular chaperones like BipA, the
heat-shock protein family of chaperones
(Hsp104, Hsp70, Hsp90) and the protein
disulfide-isomerase PdiA, thus satisfying the
increased demand for folding capacity
(Zubieta et al. 2018). Persistently misfolded
proteins are deleterious to the cell and are
degraded through the ERAD (ER-associated
degradation) pathway that includes transport
to the cytoplasm and ubiquitin-mediated degradation by the proteasome (Carvalho et al.
2011).
Several strategies have been pursued to
optimize folding. These include defensive
methods, like lowering the growth temperature
or applying weaker promoters, but also potentially more awarding methods based on stimulating the folding potential via genetic
engineering of the host organism folding
machinery.
To promote heterologous protein production in A.
awamori, the UPR pathway has been constitutively
activated by overexpressing an intron-free variant of
the hac1 gene. Using this strain, production of Trametes versicolor laccase (Lcc1) was increased sevenfold
and bovine chymosin 2.8-fold (Valkonen et al. 2003).
Similarly, heterologous production of A. niger glucose
oxidase (Gox) in a T. reesei strain overexpressing
intron-free hac1 increased production 1.8-fold (Wu
et al. 2017). In comparison, overexpression of the
bipA chaperone alone in T. reesei increased production
of Gox by 1.5-fold.
Folding of proteins containing multiple
disulfide bonds may be particularly challenging
and may benefit from increased levels of the
protein disulfide isomerase PdiA. In agreement
with this, production of thaumatin, a sweettasting plant protein containing eight disulfide
bonds, was increased twofold by using an A.
awamori strain overexpressing pdiA (Moralejo
et al. 2001). Since heterologous protein production appears to benefit from increased levels of
host chaperones, it is tempting to speculate that
more process-specific folding assistance could
be achieved by co-expressing genes encoding
one or more chaperones from the natural
source of the protein of interest.
3. Vesicular Trafficking and Polarized Growth
Filamentous fungi propagate as hyphae and the
general view is that most protein secretion
occurs at the hyphal tip (Cairns et al. 2019).
Secreted proteins are packed into vesicles,
either by cargo-receptors or as the result of
bulk flow, thereby mediating transport from
the ER to the Golgi and from the Golgi to the
plasma membrane (Barlowe and Miller 2013).
Most vesicles accumulate at the Spitzenko ¨rper
at the apex of the hyphae before fusing to the
membrane in a process that depends on the
exocyst octamer (Ahmed et al. 2018; Riquelme
and Sa ´nchez-Leo ´n 2014). Since proteins are
synthesized throughout the hyphae, vesicles
are transported by an elaborate transport system based on actin filaments and microtubules
to ensure efficient transport to the hyphal apex
(Steinberg et al. 2017). Many of the individual
steps in the secretory pathway are understood
in molecular detail, and a popular strategy aiming at enhancing secretory transport is to overexpress genes encoding proteins that are
directly involved in transport. For example,
loading of cargo proteins into vesicles and targeting vesicles to a destination membrane have
been engineered to increase secretion as exemplified below.
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
247