scripts obtained at six different growth conditions (Sibthorp et al. 2013), and this will facilitate extraction of new A. nidulans promoters as
the map simplifies promoter sequence identification.
2. Synthetic Promoters and Gene-Expression
Systems
Natural promoters are often influenced by the
state of metabolism of the host, and this may be
undesirable for heterologous production. Synthetic biology-based approaches are therefore
adopted to generate artificial/synthetic geneexpression systems that ideally act independent of the host metabolism.
For example, Gressler et al. developed an expression
system based on a fusion of the maltose-inducible
amyB promoter from A. oryzae with the ORF encoding
the transcriptional activator TerR from the Aspergillus
terreus terrein gene cluster (Gressler et al. 2015). In
addition, the system contains an expression cassette
with a bidirectional promoter that binds TerR. Subsequently, the system was used to heterologously express
two GOIs in A. niger by addition of maltose.
Another synthetic expression system was developed in
T. reesei to facilitate cellulose degradation. Cre1 and
Ace1; two general glucose stimulated repressors of
genes involved in production of cellulolytic enzymes
were fused to the activation domain of herpes simplex
virus protein 16 (VP16), hence turning glucose repression into gene activation (Zhang et al. 2018).
More advanced orthogonal systems based
on synthetic transcriptional regulators and
matching synthetic promoter sequences have
made gene expression much more controllable.
For example, synthetic biology based methods
have elegantly been applied to develop a constitutive expression system, which is almost
equally functional in distantly related fungi
including A. niger, T. reesei, and several yeasts
(Rantasalo et al. 2018). Such synthetic tools
bypass the organism specificity of conventional promoters and may become a valuable
bio-block for multi-species studies. This system
consists of a synthetic transcription factor
(sTF) composed of the LexA repressor from E.
coli fused to the activation domain from VP16.
Expression of sTF is controlled by promoters
with different arrays of LexA binding sites positioned upstream of a fungal core promoter,
which was selected for functionality in several
species. Low constitutive expression of the gene
encoding the synthetic transcription factor is
ensured via another universally active fungal
core promoter. Importantly, promoter strength
can be regulated by varying the number of LexA
binding sites in the synthetic promoter. The
system was recently combined with CRISPRCas9 multiplexing technology enabling triple
targeted integration of the GOI cassette in
three different loci in T. reesei (Rantasalo et al.
2019).
Orthogonal inducible expression platforms
based on the Tet-on and Tet-off systems have
been implemented in fungi (Fig. 10.4) (Meyer
et al. 2011; Wanka et al. 2016). The Tet-off
system is based on the synthetic transcription
factor (tTA) a fusion of the E. coli repressor
TetR with the activation domain of VP16 from
herpes simplex virus, which binds to tetO operator elements in the absence of the tetracycline
derivative doxycycline, Dox (Gossen and
Bujard 1992). In contrast, the Tet-on system is
based on rtTA-M2 (or other variants of rtTA)
(Gossen et al. 1995; Urlinger et al. 2000), which
are mutated versions of tTA that binds to tetO
operator elements in the presence of Dox. A
Dox responding synthetic promoter is made
by positioning tetO elements upstream of a
core promoter, which provides low or undetectable basal expression level in the absence of the
synthetic transcription factors tTA and rtTAM2 (Wanka et al. 2016). In the Tet-on system,
which constitutively produces rtTA-M2, the
promoter is activated by addition of Dox,
whereas in the Tet-off system, which constitutively produces tTA, the promoter is activated
in the absence of Dox. Note that the Tet-on
system is often preferred over Tet-off due to
its faster response time (Wanka et al. 2016).
D. Terminators
Terminators coordinate transcription termination and the extent of polyadenylation at the
3
0 -end of the new transcript, which in turn is
important for its nuclear export and stability
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
241
the map simplifies promoter sequence identification.
2. Synthetic Promoters and Gene-Expression
Systems
Natural promoters are often influenced by the
state of metabolism of the host, and this may be
undesirable for heterologous production. Synthetic biology-based approaches are therefore
adopted to generate artificial/synthetic geneexpression systems that ideally act independent of the host metabolism.
For example, Gressler et al. developed an expression
system based on a fusion of the maltose-inducible
amyB promoter from A. oryzae with the ORF encoding
the transcriptional activator TerR from the Aspergillus
terreus terrein gene cluster (Gressler et al. 2015). In
addition, the system contains an expression cassette
with a bidirectional promoter that binds TerR. Subsequently, the system was used to heterologously express
two GOIs in A. niger by addition of maltose.
Another synthetic expression system was developed in
T. reesei to facilitate cellulose degradation. Cre1 and
Ace1; two general glucose stimulated repressors of
genes involved in production of cellulolytic enzymes
were fused to the activation domain of herpes simplex
virus protein 16 (VP16), hence turning glucose repression into gene activation (Zhang et al. 2018).
More advanced orthogonal systems based
on synthetic transcriptional regulators and
matching synthetic promoter sequences have
made gene expression much more controllable.
For example, synthetic biology based methods
have elegantly been applied to develop a constitutive expression system, which is almost
equally functional in distantly related fungi
including A. niger, T. reesei, and several yeasts
(Rantasalo et al. 2018). Such synthetic tools
bypass the organism specificity of conventional promoters and may become a valuable
bio-block for multi-species studies. This system
consists of a synthetic transcription factor
(sTF) composed of the LexA repressor from E.
coli fused to the activation domain from VP16.
Expression of sTF is controlled by promoters
with different arrays of LexA binding sites positioned upstream of a fungal core promoter,
which was selected for functionality in several
species. Low constitutive expression of the gene
encoding the synthetic transcription factor is
ensured via another universally active fungal
core promoter. Importantly, promoter strength
can be regulated by varying the number of LexA
binding sites in the synthetic promoter. The
system was recently combined with CRISPRCas9 multiplexing technology enabling triple
targeted integration of the GOI cassette in
three different loci in T. reesei (Rantasalo et al.
2019).
Orthogonal inducible expression platforms
based on the Tet-on and Tet-off systems have
been implemented in fungi (Fig. 10.4) (Meyer
et al. 2011; Wanka et al. 2016). The Tet-off
system is based on the synthetic transcription
factor (tTA) a fusion of the E. coli repressor
TetR with the activation domain of VP16 from
herpes simplex virus, which binds to tetO operator elements in the absence of the tetracycline
derivative doxycycline, Dox (Gossen and
Bujard 1992). In contrast, the Tet-on system is
based on rtTA-M2 (or other variants of rtTA)
(Gossen et al. 1995; Urlinger et al. 2000), which
are mutated versions of tTA that binds to tetO
operator elements in the presence of Dox. A
Dox responding synthetic promoter is made
by positioning tetO elements upstream of a
core promoter, which provides low or undetectable basal expression level in the absence of the
synthetic transcription factors tTA and rtTAM2 (Wanka et al. 2016). In the Tet-on system,
which constitutively produces rtTA-M2, the
promoter is activated by addition of Dox,
whereas in the Tet-off system, which constitutively produces tTA, the promoter is activated
in the absence of Dox. Note that the Tet-on
system is often preferred over Tet-off due to
its faster response time (Wanka et al. 2016).
D. Terminators
Terminators coordinate transcription termination and the extent of polyadenylation at the
3
0 -end of the new transcript, which in turn is
important for its nuclear export and stability
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
241