To stimulate secretion of a heterologous protein, the
vesicular transport was enhanced by overexpressing
rabD, encoding a Rab GTPase involved in transport
of exocytic post-Golgi vesicles (Pantazopoulou et al.
2014), the deletion of which reduces protein secretion
(Punt et al. 2001). Specifically, a fusion of carrier glucoamylase to mRFP via a Kex2 cleavable linker (Kex2cl)
increased production of mRFP in A. nidulans by 40%
(Schale ´n et al. 2016) in the rabD overexpression background. Hoang and co-workers showed that Vip36, a
putative lectin-type cargo receptor inferred to be
involved in vesicular cargo loading of glycoproteins
between ER and Golgi, facilitates secretion of the
recombinant fusion proteins AmyB-Kex2cl-GFP and
AmyB-Kex2cl-chymosin in A. oryzae, presumably by
reducing protein retention in the ER (Hoang et al.
2015). Importantly, overexpression of vip36 in strains
producing the recombinant GFP fusion protein led to
sevenfold increase in the levels of extracellular GFP
(Hoang et al. 2015). Finally, docking of vesicles to a
target membrane was stimulated by overexpressing
snc1 encoding a vSNARE, and this feature increased
secretion of A. niger glucose oxidase in T. reesei by
2.2-fold (Wu et al. 2017).
Similarly, specific genes involved in the
secretory pathway can be deleted to positively
influence heterologous protein production. For
example, the vacuolar protein sorting receptor
Vps10 mediates transport of proteins from the
Golgi to the vacuoles; and it has been observed
that heterologous proteins designated for secretion partially accumulate in the vacuoles (Masai
et al. 2003). This principle has been exploited in
A. oryzae where a deletion of vps10 improved
production of recombinant human lysosome
and bovine chymosin 2.2- and 3-fold, respectively (Yoon et al. 2010). Even the micromorphology of the mycelium can be manipulated to
enhance protein secretion. In an elegant study,
Fiedler et al. produced a hyperbranching A.
niger strain by deleting the gene encoding the
Rho G-protein RacA, a key protein in maintaining polarized growth (Kwon et al. 2011); and
this feature increased glucoamylase production
four-fold (Fiedler et al. 2018).
C. Building Blocks for Protein Synthesis
Many filamentous fungi secrete large amounts
of enzymes and production of these proteins
consume building blocks that could be used
for the desired heterologous protein. Hence,
additional building blocks for heterologous
protein production can be obtained by deleting
genes encoding the most abundant secreted
proteins. This strategy has the further advantages that it reduces pressure on the secretory
pathway machinery and that subsequent product purification is simplified. In its simplest
scheme, this approach can be utilized by deleting genes encoding major secreted products,
such as a-amylase A and B of A. oryzae, thereby
providing a strain with no detectable a-amylase
activity (Kitamoto et al. 2015). Toward the same
goal, genes encoding transcriptional activators
of major cellulolytic enzymes have been deleted
in both A. niger and T. reesei.
In A. niger, deletion of amyR reduced the total amount
of secreted protein 16.4-fold (Zhang et al. 2016b). Likewise, deletion of xyr1 in T. reesei abolished expression
of the genes cbhI and cbhII encoding the major cellobiohydrolases CBHI and CBHII, which may constitute
up to 80% of the total amount of secreted protein
(Bergquist et al. 2004; Stricker et al. 2006).
While these approaches diminished expression of endogenous enzymes, it may also be
advantageous to modify the GOI expression
cassette. To this end, for driving secretion of a
heterologously produced protein, Rantasalo
et al. exchanged usage of a carrier protein for
a smaller secretion signal, thereby reducing the
total secreted protein twofold and inferring a
substantial increase in recombinant enzyme
purity (Rantasalo et al. 2019).
Comparative transcriptomics approaches
have been used to uncover engineering targets
that enhance heterologous protein production.
For example, processes related to biosynthesis
of amino acids and tRNAs are upregulated in
strains of A. nidulans overexpressing heterologous enzymes (Zubieta et al. 2018) and in A.
niger CBS 513.88, a classic enzyme production
strain (Andersen et al. 2011). Hence, building
block availability may be a limiting factor that
could be improved by genetic engineering, e.g.,
by increasing expression of genes encoding
specific transporters or genes required for
making aminoacryl-tRNAs.
248
J. K. H. Rendsvig et al.
vesicular transport was enhanced by overexpressing
rabD, encoding a Rab GTPase involved in transport
of exocytic post-Golgi vesicles (Pantazopoulou et al.
2014), the deletion of which reduces protein secretion
(Punt et al. 2001). Specifically, a fusion of carrier glucoamylase to mRFP via a Kex2 cleavable linker (Kex2cl)
increased production of mRFP in A. nidulans by 40%
(Schale ´n et al. 2016) in the rabD overexpression background. Hoang and co-workers showed that Vip36, a
putative lectin-type cargo receptor inferred to be
involved in vesicular cargo loading of glycoproteins
between ER and Golgi, facilitates secretion of the
recombinant fusion proteins AmyB-Kex2cl-GFP and
AmyB-Kex2cl-chymosin in A. oryzae, presumably by
reducing protein retention in the ER (Hoang et al.
2015). Importantly, overexpression of vip36 in strains
producing the recombinant GFP fusion protein led to
sevenfold increase in the levels of extracellular GFP
(Hoang et al. 2015). Finally, docking of vesicles to a
target membrane was stimulated by overexpressing
snc1 encoding a vSNARE, and this feature increased
secretion of A. niger glucose oxidase in T. reesei by
2.2-fold (Wu et al. 2017).
Similarly, specific genes involved in the
secretory pathway can be deleted to positively
influence heterologous protein production. For
example, the vacuolar protein sorting receptor
Vps10 mediates transport of proteins from the
Golgi to the vacuoles; and it has been observed
that heterologous proteins designated for secretion partially accumulate in the vacuoles (Masai
et al. 2003). This principle has been exploited in
A. oryzae where a deletion of vps10 improved
production of recombinant human lysosome
and bovine chymosin 2.2- and 3-fold, respectively (Yoon et al. 2010). Even the micromorphology of the mycelium can be manipulated to
enhance protein secretion. In an elegant study,
Fiedler et al. produced a hyperbranching A.
niger strain by deleting the gene encoding the
Rho G-protein RacA, a key protein in maintaining polarized growth (Kwon et al. 2011); and
this feature increased glucoamylase production
four-fold (Fiedler et al. 2018).
C. Building Blocks for Protein Synthesis
Many filamentous fungi secrete large amounts
of enzymes and production of these proteins
consume building blocks that could be used
for the desired heterologous protein. Hence,
additional building blocks for heterologous
protein production can be obtained by deleting
genes encoding the most abundant secreted
proteins. This strategy has the further advantages that it reduces pressure on the secretory
pathway machinery and that subsequent product purification is simplified. In its simplest
scheme, this approach can be utilized by deleting genes encoding major secreted products,
such as a-amylase A and B of A. oryzae, thereby
providing a strain with no detectable a-amylase
activity (Kitamoto et al. 2015). Toward the same
goal, genes encoding transcriptional activators
of major cellulolytic enzymes have been deleted
in both A. niger and T. reesei.
In A. niger, deletion of amyR reduced the total amount
of secreted protein 16.4-fold (Zhang et al. 2016b). Likewise, deletion of xyr1 in T. reesei abolished expression
of the genes cbhI and cbhII encoding the major cellobiohydrolases CBHI and CBHII, which may constitute
up to 80% of the total amount of secreted protein
(Bergquist et al. 2004; Stricker et al. 2006).
While these approaches diminished expression of endogenous enzymes, it may also be
advantageous to modify the GOI expression
cassette. To this end, for driving secretion of a
heterologously produced protein, Rantasalo
et al. exchanged usage of a carrier protein for
a smaller secretion signal, thereby reducing the
total secreted protein twofold and inferring a
substantial increase in recombinant enzyme
purity (Rantasalo et al. 2019).
Comparative transcriptomics approaches
have been used to uncover engineering targets
that enhance heterologous protein production.
For example, processes related to biosynthesis
of amino acids and tRNAs are upregulated in
strains of A. nidulans overexpressing heterologous enzymes (Zubieta et al. 2018) and in A.
niger CBS 513.88, a classic enzyme production
strain (Andersen et al. 2011). Hence, building
block availability may be a limiting factor that
could be improved by genetic engineering, e.g.,
by increasing expression of genes encoding
specific transporters or genes required for
making aminoacryl-tRNAs.
248
J. K. H. Rendsvig et al.