inefficient when suboptimal signals are applied.
Pre-sequences encode secretion signals that
mediate protein translocation into the ER,
while pro-sequences may facilitate the folding
process and maintain proteins as inactive
forms until its removal. One of the three general
approaches is typically used to ensure secretion
of a heterologous protein. In the first approach,
secretion of the recombinant protein simply
relies on the secretion signals of the native
protein. In the second approach, the Nterminus of the recombinant protein is
extended with either a pre- or pre-prosequence from a highly secreted protein of the
production host (or from a closely related species) or thirdly N-terminal extension with a
secreted carrier protein.
The first, and most simple, approach
employs the native secretion signal of the heterologous protein. However, the fact that the
sorting signal are from a different organism
raises questions concerning their ability to support efficient secretion in the host. The observation that heterologous proteins originating
from species closely related to the host often
sort quite efficiently suggests that their secretion signals are functional.
For example, glucoamylase (glaA) from A. niger and
rhamnogalacturonate lyase A (rglA) from Aspergillus
sojae have been efficiently produced via their own
secretion signals in A. nidulans (Schale ´n et al. 2016)
and in A. oryzae (Yoshino-Yasuda et al. 2012), respectively.
Secretion signals may also be functional
between more distantly related fungi, as
observed with two basidiomycete laccases,
Lcc1 of Pycnoporus coccineus and Lcc of Pycnoporus sanguineus, successfully secreted by the
hosts A. oryzae and A. nidulans using the proteins native secretion signals, albeit with low
yields (Hoshida et al. 2005; Li et al. 2018).
More efficient secretion may be achieved by
using host endogenous signals as they are
expected to be more proficiently recognized
and processed by the host. The second
approach therefore employs the pre- or prepro-sequence of a highly secreted protein originating from the host, or a closely related
species, to enhance secretion of the heterologous protein.
A successful example of this approach is production of
basidiomycete laccase, Lac1, from Pycnoporus cinnabarinus in A. niger (Record et al. 2002). In this study,
an 80-fold increase in extracellular activity was
achieved by replacing the natural pre-sequence of
Lac1 with the pre-pro-sequence of A. niger glaA.
In the final approach, a carrier protein is
fused to the heterologous protein to improve
production by alleviating post-translational
bottlenecks and, in some cases, by increasing
mRNA levels (Gouka et al. 1997). Comparative
DNA microarrays of A. oryzae strains expressing heterologous bovine chymosin, with and
without a carrier protein (AmyB), showed that
inclusion of the carrier protein promoted
induction of the UPR (see Sect. IV.A.2.b) and
increased expression of genes encoding secretory chaperones and proteins involved in intracellular trafficking, thereby facilitating folding
and secretion (Ohno et al. 2011). Examples of
proteins that have been used as carriers are
GlaA, AmyB, and CbhI from A. niger, A. oryzae,
and T. reesei, respectively. To liberate the heterologous protein from the carrier protein
in vivo, the two proteins are fused via a linker
containing a Kex2 cleavage site (KR/RR) to
enable proteolytic separation in the late-Golgi
(Jin et al. 2007; Landowski et al. 2016).
Employing this approach, Gouka et al. used A. niger
GlaA as carrier protein for heterologous production of
human interleukin-6 in Aspergillus awamori, thereby
improving the extracellular protein yield 100-fold
(Gouka et al. 1997).
In an interesting variant of this approach,
Jin et al. produced human lysozyme in A. oryzae using a gene cassette containing the amyB
gene fused to tandem gene copies of HLY
encoding lysosome, which were separated by
sequences encoding Kex2 cleavage sites (Jin
et al. 2007). However, releasing the heterologous protein from the carrier protein may be a
bottleneck. For example, during production of
human interferon (IFNa-2b) in T. reesei using
Cbh1 as a carrier protein, 44% of the fusion
protein was not cleaved by Kex2 (Landowski
et al. 2016). More efficient Kex2 cleavage has
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
245
Pre-sequences encode secretion signals that
mediate protein translocation into the ER,
while pro-sequences may facilitate the folding
process and maintain proteins as inactive
forms until its removal. One of the three general
approaches is typically used to ensure secretion
of a heterologous protein. In the first approach,
secretion of the recombinant protein simply
relies on the secretion signals of the native
protein. In the second approach, the Nterminus of the recombinant protein is
extended with either a pre- or pre-prosequence from a highly secreted protein of the
production host (or from a closely related species) or thirdly N-terminal extension with a
secreted carrier protein.
The first, and most simple, approach
employs the native secretion signal of the heterologous protein. However, the fact that the
sorting signal are from a different organism
raises questions concerning their ability to support efficient secretion in the host. The observation that heterologous proteins originating
from species closely related to the host often
sort quite efficiently suggests that their secretion signals are functional.
For example, glucoamylase (glaA) from A. niger and
rhamnogalacturonate lyase A (rglA) from Aspergillus
sojae have been efficiently produced via their own
secretion signals in A. nidulans (Schale ´n et al. 2016)
and in A. oryzae (Yoshino-Yasuda et al. 2012), respectively.
Secretion signals may also be functional
between more distantly related fungi, as
observed with two basidiomycete laccases,
Lcc1 of Pycnoporus coccineus and Lcc of Pycnoporus sanguineus, successfully secreted by the
hosts A. oryzae and A. nidulans using the proteins native secretion signals, albeit with low
yields (Hoshida et al. 2005; Li et al. 2018).
More efficient secretion may be achieved by
using host endogenous signals as they are
expected to be more proficiently recognized
and processed by the host. The second
approach therefore employs the pre- or prepro-sequence of a highly secreted protein originating from the host, or a closely related
species, to enhance secretion of the heterologous protein.
A successful example of this approach is production of
basidiomycete laccase, Lac1, from Pycnoporus cinnabarinus in A. niger (Record et al. 2002). In this study,
an 80-fold increase in extracellular activity was
achieved by replacing the natural pre-sequence of
Lac1 with the pre-pro-sequence of A. niger glaA.
In the final approach, a carrier protein is
fused to the heterologous protein to improve
production by alleviating post-translational
bottlenecks and, in some cases, by increasing
mRNA levels (Gouka et al. 1997). Comparative
DNA microarrays of A. oryzae strains expressing heterologous bovine chymosin, with and
without a carrier protein (AmyB), showed that
inclusion of the carrier protein promoted
induction of the UPR (see Sect. IV.A.2.b) and
increased expression of genes encoding secretory chaperones and proteins involved in intracellular trafficking, thereby facilitating folding
and secretion (Ohno et al. 2011). Examples of
proteins that have been used as carriers are
GlaA, AmyB, and CbhI from A. niger, A. oryzae,
and T. reesei, respectively. To liberate the heterologous protein from the carrier protein
in vivo, the two proteins are fused via a linker
containing a Kex2 cleavage site (KR/RR) to
enable proteolytic separation in the late-Golgi
(Jin et al. 2007; Landowski et al. 2016).
Employing this approach, Gouka et al. used A. niger
GlaA as carrier protein for heterologous production of
human interleukin-6 in Aspergillus awamori, thereby
improving the extracellular protein yield 100-fold
(Gouka et al. 1997).
In an interesting variant of this approach,
Jin et al. produced human lysozyme in A. oryzae using a gene cassette containing the amyB
gene fused to tandem gene copies of HLY
encoding lysosome, which were separated by
sequences encoding Kex2 cleavage sites (Jin
et al. 2007). However, releasing the heterologous protein from the carrier protein may be a
bottleneck. For example, during production of
human interferon (IFNa-2b) in T. reesei using
Cbh1 as a carrier protein, 44% of the fusion
protein was not cleaved by Kex2 (Landowski
et al. 2016). More efficient Kex2 cleavage has
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
245