been achieved by overexpressing kex2 (Landowski et al. 2016) or by optimizing Kex2 cleavage sites (Lin et al. 2006; Nakajima et al. 2006;
Yang et al. 2013), e.g., by using the entire prosequence of GlaA, NVISKR, as a Kex2 cleavage
site (Landowski et al. 2016).
It is important to stress that the choice of
optimal signal sequences is not straightforward. For example, Rantasalo et al. optimized
secretion of lipase B from Candida antarctica
(CalB) in T. reesei (Rantasalo et al. 2019) by
using natural secretion signals of the host proteins CbhI and CbhII and secretion signals from
heterologous AmyA and GlaA from A. awamori
and A. niger, respectively, and by using CbhI as
a carrier protein. When the lipase yields
obtained with the different strains were compared, the strain employing the secretion signal
of AmyA performed best. On the other hand,
studies on heterologous production of Cbh1 in
A. awamori using different secretion signals
showed no differences illustrating that gains
may be dependent on the heterologous protein
and/or the host (Adney et al. 2003; Chou et al.
2004).
2. Glycosylation and Folding
Glycosylation of the heterologous protein is
important for the subsequent folding, and without validity of these properties, the protein
activity may be impaired or abolished. In the
heterologous production host, these processes
may be executed in alternate fashion compared
to the native producer, leading to reduced
yields or non-functional proteins. Therefore,
substantial efforts have been conducted to
improve both processes by increasing or reducing expression of genes encoding proteins
involved in the glycosylation and folding
machinery of the production host to enhance
protein production.
a) Glycosylation
Protein glycosylation has multifaceted impact
on heterologous protein production. Firstly, it
affects protein folding and secretion efficiencies; secondly, it influences the final properties
of the enzyme including its stability, solubility,
and catalytic parameters; and finally, it acts to
protect the enzymes from proteases (Gupta and
Shukla 2018; Li and d’Anjou 2009). In agreement with this view, mutation of the four Nglycosylation sites of A. terreus b-glucosidase
resulted in reduced thermal stability and 15–
35% decreased specific activity and catalytic
rate as compared to the wild-type variant
when heterologously produced by T. reesei
(Wei et al. 2013). Hence, proper glycosylation
is often required to achieve high production
yields. For example, analysis of bovine chymosin produced by A. niger showed that an Nglycosylation site was poorly glycosylated by
this host. Importantly, the extracellular activity
yields could be increased by up to 100% in A.
niger by optimizing the glycosylation efficiency of this site by site-directed mutagenesis
(van den Brink et al. 2006). Similarly, production yields and quality may also be increased by
engineering entirely new glycosylation sites
into proteins. Using this approach, heterologous production activity yields of cellobiohydrolase Cel7A from Penicillium funiculosum in
A. awamori were increased by 70% by engineering a novel N-glycosylation motif into the
protein at position N194 (Adney et al. 2009). On
the other hand, production yields and quality
may be decreased if non-native glycosylation
sites are unintentionally or aberrantly glycosylated by the glycosylation machinery of the
heterologous host. Hence, a 70% increase of T.
reesei Cel7A activity yields were achieved by
removing a glycosylation site (N384), which
contained a larger glycan structure when using
A. awamori as production host, than in the
native organism T. reesei (Adney et al. 2009).
Fungi may also potentially be used as hosts for
production of therapeutic proteins. The fact
that different species add different sugar structures to proteins poses an additional production challenge, as therapeutic proteins
containing aberrant sugar moieties may cause
immune responses in patients. In yeasts, this
challenge has been successfully addressed by
humanizing the glycosylation pathway by
genetic engineering (Gupta and Shukla 2018)
indicating that production of therapeutic pro246
J. K. H. Rendsvig et al.
Yang et al. 2013), e.g., by using the entire prosequence of GlaA, NVISKR, as a Kex2 cleavage
site (Landowski et al. 2016).
It is important to stress that the choice of
optimal signal sequences is not straightforward. For example, Rantasalo et al. optimized
secretion of lipase B from Candida antarctica
(CalB) in T. reesei (Rantasalo et al. 2019) by
using natural secretion signals of the host proteins CbhI and CbhII and secretion signals from
heterologous AmyA and GlaA from A. awamori
and A. niger, respectively, and by using CbhI as
a carrier protein. When the lipase yields
obtained with the different strains were compared, the strain employing the secretion signal
of AmyA performed best. On the other hand,
studies on heterologous production of Cbh1 in
A. awamori using different secretion signals
showed no differences illustrating that gains
may be dependent on the heterologous protein
and/or the host (Adney et al. 2003; Chou et al.
2004).
2. Glycosylation and Folding
Glycosylation of the heterologous protein is
important for the subsequent folding, and without validity of these properties, the protein
activity may be impaired or abolished. In the
heterologous production host, these processes
may be executed in alternate fashion compared
to the native producer, leading to reduced
yields or non-functional proteins. Therefore,
substantial efforts have been conducted to
improve both processes by increasing or reducing expression of genes encoding proteins
involved in the glycosylation and folding
machinery of the production host to enhance
protein production.
a) Glycosylation
Protein glycosylation has multifaceted impact
on heterologous protein production. Firstly, it
affects protein folding and secretion efficiencies; secondly, it influences the final properties
of the enzyme including its stability, solubility,
and catalytic parameters; and finally, it acts to
protect the enzymes from proteases (Gupta and
Shukla 2018; Li and d’Anjou 2009). In agreement with this view, mutation of the four Nglycosylation sites of A. terreus b-glucosidase
resulted in reduced thermal stability and 15–
35% decreased specific activity and catalytic
rate as compared to the wild-type variant
when heterologously produced by T. reesei
(Wei et al. 2013). Hence, proper glycosylation
is often required to achieve high production
yields. For example, analysis of bovine chymosin produced by A. niger showed that an Nglycosylation site was poorly glycosylated by
this host. Importantly, the extracellular activity
yields could be increased by up to 100% in A.
niger by optimizing the glycosylation efficiency of this site by site-directed mutagenesis
(van den Brink et al. 2006). Similarly, production yields and quality may also be increased by
engineering entirely new glycosylation sites
into proteins. Using this approach, heterologous production activity yields of cellobiohydrolase Cel7A from Penicillium funiculosum in
A. awamori were increased by 70% by engineering a novel N-glycosylation motif into the
protein at position N194 (Adney et al. 2009). On
the other hand, production yields and quality
may be decreased if non-native glycosylation
sites are unintentionally or aberrantly glycosylated by the glycosylation machinery of the
heterologous host. Hence, a 70% increase of T.
reesei Cel7A activity yields were achieved by
removing a glycosylation site (N384), which
contained a larger glycan structure when using
A. awamori as production host, than in the
native organism T. reesei (Adney et al. 2009).
Fungi may also potentially be used as hosts for
production of therapeutic proteins. The fact
that different species add different sugar structures to proteins poses an additional production challenge, as therapeutic proteins
containing aberrant sugar moieties may cause
immune responses in patients. In yeasts, this
challenge has been successfully addressed by
humanizing the glycosylation pathway by
genetic engineering (Gupta and Shukla 2018)
indicating that production of therapeutic pro246
J. K. H. Rendsvig et al.