“cleavage” at the 2A spacer results in a partial
2A peptide sequence remaining at the Cterminus of the protein, which may interfere
with protein function and therefore needs to
be removed, e.g., by combinatory usage with
other proteolytic sites (Hoefgen et al. 2018).
Moreover, as “cleavage” at the 2A spacer is
not 100%, the relative efficiency of protein production often depends on the position of the
proteins ORF in the transcript (Schuetze and
Meyer 2017).
Lastly, we note that for all strategies, two
genes can be inserted simultaneously as a single
cassette if bidirectional promoters (Wiemann
et al. 2018; Rendsvig et al. 2019) are used to
control gene expression of the heterologous
gene pairs. This feature can be used to speed
up strain construction.
III. Bio-Blocks
A bio-block is a DNA sequence that encompasses a molecularly functional unit. This
includes nucleotide sequences, e.g., promoters
and terminators, or sequences encoding a protein, e.g., the GOI and selection marker, or
shorter protein sequences such as secretion
signals and purification tags. Generation of the
basic gene-expression cassette for production
of a heterologous protein requires several bioblocks, including the GOI, promoter, and terminator. Selection markers may also be
included in the construct, offering versatile
applications in different scenarios. In recent
years, the use of -omics data has driven the
discovery of natural promoters, which are
active during the desired cultivation conditions
for production. While the majority of strain
engineering has been based on natural genetic
elements for controlling gene expression, significant headway has been made in the development of synthetic promoters and
terminators. In extension, even full synthetic
gene-expression systems have been established, in which several individual genetic elements (natural or modified) are combined, to
facilitate controlled gene expression and
enhance productivity. Moreover, to enable
secretion of the protein, a transport signal
must be included as an additional bio-block
(Sect. IV.A.1). For specialized purposes like
protein visualization or purification, protein
tags fulfilling such tasks are also included in
the construct, thereby increasing the pool of
bio-blocks.
A. Gene of Interest
Since the goal of heterologous expression is to
produce products derived from one or more
genes of interest, this element can also be considered the most important bio-block. As the
GOIs coding potential in most cases should not
be changed, it therefore constitutes the least
flexible bio-block; however, its design requires
several considerations. The GOI sequence may
be derived from different sources. In the simplest scheme, if the donor strain is related to the
new host, genomic DNA can typically serve as a
template to make a functional PCR-derived GOI
bio-block. However, if the donor strain is more
distantly related, intron splicing and codon
bias may compromise gene expression and
translation, and we will briefly review the two
latter issues below.
1. Introns
The presence of introns in a heterologous gene
may result in reduced mRNA production due to
mis- and incomplete splicing (He and Cox
2016; Zhao et al. 2013). In principle, the problem can be solved by employing cDNA as a PCR
template for making the GOI bio-block or alternatively fuse PCR-derived exons by another
round of PCR or by a one-step cloning method
that allows for seamless assembly of multiple
fragments (An et al. 2007). Alternatively, an
entirely synthetic gene may be acquired from
a commercial source. However, transcription,
splicing, polyadenylation, and mRNA export
are a coordinated process (Bentley 2014) and
as a result the presence of introns may affect
gene expression.
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
233
2A peptide sequence remaining at the Cterminus of the protein, which may interfere
with protein function and therefore needs to
be removed, e.g., by combinatory usage with
other proteolytic sites (Hoefgen et al. 2018).
Moreover, as “cleavage” at the 2A spacer is
not 100%, the relative efficiency of protein production often depends on the position of the
proteins ORF in the transcript (Schuetze and
Meyer 2017).
Lastly, we note that for all strategies, two
genes can be inserted simultaneously as a single
cassette if bidirectional promoters (Wiemann
et al. 2018; Rendsvig et al. 2019) are used to
control gene expression of the heterologous
gene pairs. This feature can be used to speed
up strain construction.
III. Bio-Blocks
A bio-block is a DNA sequence that encompasses a molecularly functional unit. This
includes nucleotide sequences, e.g., promoters
and terminators, or sequences encoding a protein, e.g., the GOI and selection marker, or
shorter protein sequences such as secretion
signals and purification tags. Generation of the
basic gene-expression cassette for production
of a heterologous protein requires several bioblocks, including the GOI, promoter, and terminator. Selection markers may also be
included in the construct, offering versatile
applications in different scenarios. In recent
years, the use of -omics data has driven the
discovery of natural promoters, which are
active during the desired cultivation conditions
for production. While the majority of strain
engineering has been based on natural genetic
elements for controlling gene expression, significant headway has been made in the development of synthetic promoters and
terminators. In extension, even full synthetic
gene-expression systems have been established, in which several individual genetic elements (natural or modified) are combined, to
facilitate controlled gene expression and
enhance productivity. Moreover, to enable
secretion of the protein, a transport signal
must be included as an additional bio-block
(Sect. IV.A.1). For specialized purposes like
protein visualization or purification, protein
tags fulfilling such tasks are also included in
the construct, thereby increasing the pool of
bio-blocks.
A. Gene of Interest
Since the goal of heterologous expression is to
produce products derived from one or more
genes of interest, this element can also be considered the most important bio-block. As the
GOIs coding potential in most cases should not
be changed, it therefore constitutes the least
flexible bio-block; however, its design requires
several considerations. The GOI sequence may
be derived from different sources. In the simplest scheme, if the donor strain is related to the
new host, genomic DNA can typically serve as a
template to make a functional PCR-derived GOI
bio-block. However, if the donor strain is more
distantly related, intron splicing and codon
bias may compromise gene expression and
translation, and we will briefly review the two
latter issues below.
1. Introns
The presence of introns in a heterologous gene
may result in reduced mRNA production due to
mis- and incomplete splicing (He and Cox
2016; Zhao et al. 2013). In principle, the problem can be solved by employing cDNA as a PCR
template for making the GOI bio-block or alternatively fuse PCR-derived exons by another
round of PCR or by a one-step cloning method
that allows for seamless assembly of multiple
fragments (An et al. 2007). Alternatively, an
entirely synthetic gene may be acquired from
a commercial source. However, transcription,
splicing, polyadenylation, and mRNA export
are a coordinated process (Bentley 2014) and
as a result the presence of introns may affect
gene expression.
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
233