D. Protease-Deficient Strains
The saprophytic lifestyle of filamentous fungi
is key to their high secretion capacity. In addition to facilitating heterologous protein production, a consequence of this lifestyle is
secretion of large amounts of proteases,
which the fungi use for extracellular biomass
degradation. For example, proteomic analysis
of the T. reesei QM6a secretome under
submerged cultivation showed pH-dependent
secretion of 39 peptidases and proteinases,
with more than 20 secreted simultaneously
(Adav et al. 2011). Since these proteases need
to digest a broad spectrum of substrates,
chances are that the heterologous protein is
also degraded leading to decreasing activity of
the recombinant protein over time (Kamaruddin et al. 2018). In addition, intracellular proteases in the secretory pathway, such as Kex2,
may also degrade recombinant proteins, which
can be resolved by mutagenizing the cleavage
site (Lin et al. 2006). The fact that addition of
protease inhibitors, such as trypsin and aspartic protease inhibitor (Landowski et al. 2015),
or use of strains with different protease profiles (Sun et al. 2016) can lead to significantly
higher protein yields suggests that a strategy
aiming at producing protease-deficient
strains could be advantageous. However,
whereas elimination of some proteases does
not impact fitness, some may be involved in
cell wall maintenance, and their absence often
causes an altered undesired morphology.
Hence, a gain in product yield may be lost in
growth rate or overall productivity, and the
relevant strain modifications vary from product to product even with the same host (Landowski et al. 2015).
In a simple case, Li et al. applied A. nidulans as host for
heterologous production of a P. sanguineus laccase
(Pslcc) and demonstrated that deletion of two genes
encoding major proteolytic activities, proteases
dipeptidyl-peptidase DppV and aspartic protease
PepA, increased laccase production 13-fold (Li et al.
2018).
In a more elaborate study, Kitamoto et al. generated an
A. oryzae strain with five protease gene deletions (alp1,
npI, npII, pepA, pepE) thereby reducing extracellular
protease activity to 1% (Kitamoto et al. 2015) and
providing a production strain with minimal proteolysis.
In the post-genomic era, it is possible to
make a rational gene deletion strategy based
on proteomics where abundant proteases can
be identified and linked to their genes. Using
this strategy in T. reesei, nine target genes
(pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5,
amp2) were identified and deleted to produce a
strain where the majority of proteolytic product
degradation is abolished (Landowski et al.
2015, 2016). The authors employed this strain
for production of human interferon alpha-2b
(IFNa-2b) and obtained 2.4 g/L of correctly
processed IFNa-2b.
In contrast to deleting individual protease
genes, similar effects may be achieved by RNAi
silencing of individual genes (Kitamoto et al.
2015; Qin et al. 2012). In another approach,
reduction of a set of proteases was achieved
by deleting a general transcription factor
gene, namely, prtT in A. niger, which reduced
the levels of several secreted proteases, including PepA, PepB, and PepF. After producing
Glomerella cingulata cutinase in this A. niger
strain a 25-fold increase of residual activity was
observed in culture filtrates after 2 weeks
(Kamaruddin et al. 2018).
V. Heterologous Production of
Secondary Metabolites in
Filamentous Fungi
The genome sequencing projects have uncovered a vast repertoire of fungal SMs, which
serves as an underexploited source of new
food additives and drug candidates. This process has been facilitated by the fact that all
genes required for production of a specific SM
are typically organized in a biosynthetic gene
cluster (BGC) (Keller 2019; Keller et al. 2005;
Rokas et al. 2018). However, it is difficult to
exploit the fungal SM potential for several reasons. Firstly, the vast majority of the fungal SM
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
249
The saprophytic lifestyle of filamentous fungi
is key to their high secretion capacity. In addition to facilitating heterologous protein production, a consequence of this lifestyle is
secretion of large amounts of proteases,
which the fungi use for extracellular biomass
degradation. For example, proteomic analysis
of the T. reesei QM6a secretome under
submerged cultivation showed pH-dependent
secretion of 39 peptidases and proteinases,
with more than 20 secreted simultaneously
(Adav et al. 2011). Since these proteases need
to digest a broad spectrum of substrates,
chances are that the heterologous protein is
also degraded leading to decreasing activity of
the recombinant protein over time (Kamaruddin et al. 2018). In addition, intracellular proteases in the secretory pathway, such as Kex2,
may also degrade recombinant proteins, which
can be resolved by mutagenizing the cleavage
site (Lin et al. 2006). The fact that addition of
protease inhibitors, such as trypsin and aspartic protease inhibitor (Landowski et al. 2015),
or use of strains with different protease profiles (Sun et al. 2016) can lead to significantly
higher protein yields suggests that a strategy
aiming at producing protease-deficient
strains could be advantageous. However,
whereas elimination of some proteases does
not impact fitness, some may be involved in
cell wall maintenance, and their absence often
causes an altered undesired morphology.
Hence, a gain in product yield may be lost in
growth rate or overall productivity, and the
relevant strain modifications vary from product to product even with the same host (Landowski et al. 2015).
In a simple case, Li et al. applied A. nidulans as host for
heterologous production of a P. sanguineus laccase
(Pslcc) and demonstrated that deletion of two genes
encoding major proteolytic activities, proteases
dipeptidyl-peptidase DppV and aspartic protease
PepA, increased laccase production 13-fold (Li et al.
2018).
In a more elaborate study, Kitamoto et al. generated an
A. oryzae strain with five protease gene deletions (alp1,
npI, npII, pepA, pepE) thereby reducing extracellular
protease activity to 1% (Kitamoto et al. 2015) and
providing a production strain with minimal proteolysis.
In the post-genomic era, it is possible to
make a rational gene deletion strategy based
on proteomics where abundant proteases can
be identified and linked to their genes. Using
this strategy in T. reesei, nine target genes
(pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5,
amp2) were identified and deleted to produce a
strain where the majority of proteolytic product
degradation is abolished (Landowski et al.
2015, 2016). The authors employed this strain
for production of human interferon alpha-2b
(IFNa-2b) and obtained 2.4 g/L of correctly
processed IFNa-2b.
In contrast to deleting individual protease
genes, similar effects may be achieved by RNAi
silencing of individual genes (Kitamoto et al.
2015; Qin et al. 2012). In another approach,
reduction of a set of proteases was achieved
by deleting a general transcription factor
gene, namely, prtT in A. niger, which reduced
the levels of several secreted proteases, including PepA, PepB, and PepF. After producing
Glomerella cingulata cutinase in this A. niger
strain a 25-fold increase of residual activity was
observed in culture filtrates after 2 weeks
(Kamaruddin et al. 2018).
V. Heterologous Production of
Secondary Metabolites in
Filamentous Fungi
The genome sequencing projects have uncovered a vast repertoire of fungal SMs, which
serves as an underexploited source of new
food additives and drug candidates. This process has been facilitated by the fact that all
genes required for production of a specific SM
are typically organized in a biosynthetic gene
cluster (BGC) (Keller 2019; Keller et al. 2005;
Rokas et al. 2018). However, it is difficult to
exploit the fungal SM potential for several reasons. Firstly, the vast majority of the fungal SM
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
249