differences. Hence, insertion of geneexpression cassettes into defined loci enables
comparative screening of expression levels or
enzyme activities based on different genetic
elements employed in the cassette, such as promoter, secretion signal, and terminator
sequences. Similarly, the effects achieved by
changing the GOI sequence can be directly
compared. In this way it is possible to address
whether changes in the GOI codon composition
increase yields or whether mutations infer
changes in the heterologous protein that influences its activity, specificity, folding, and/or
stability (Hansen et al. 2011b; Holm 2013).
Gene-expression cassettes can be inserted
into integration sites in the genome by HR. To
ensure high expression levels, it may be useful to
position integration sites in intergenic regions
located in transcriptionally highly active sections of a chromosome. With this method, the
gene-expression cassettes must be flanked by
up- and downstream targeting sequences matching the genomic expression site. Since fungi
rarely prefer to integrate foreign DNA into its
genome by the HR pathway, extensive screening
for the desired transformant may be necessary.
The screening workload can be reduced by using
bipartite gene-targeting substrates that select
for HR proficient protoplasts (Nielsen et al.
2006) or avoided by using NHEJ-deficient
strains (Meyer et al. 2007). Alternatively, methods based on restriction enzymes or CRISPR
technology can be employed (Ouedraogo et al.
2016; Zheng et al. 2017). Importantly with the
latter technology, GOIs can be inserted in a
marker-free manner and multiplexing is possible (Liu et al. 2015; Nødvig et al. 2018; Zhang
et al. 2016a). In fact, CRISPR mediates very
efficient gene targeting even in NHEJ-proficient
strains (Nødvig et al. 2018), eliminating the risks
of working with strains with a defective DNA
repair pathway. We therefore envision that
CRISPR-based methods will deliver the preferred tools for introducing gene-expression cassettes into defined chromosomal expression
platforms.
C. Bio-Block-Based Multi-GOI Expression
Strategies
In many cases, two or more genes are required
for the synthesis of a desired product. For
example, for heterologous production of SMs,
several genes are commonly required to synthesize the product. Above, we described that it is
possible to transfer large chromosomal fragments containing entire gene clusters from
one fungus to another (Sect. II.B.1). However,
in many cases the activation mechanism for
the genes in the cluster is not known and
may depend on unknown or even host-specific
transcription factors (TF) (Keller 2019); see
Chap. 11. Moreover, not all genes contributing
to the biosynthetic pathway of interest may be
situated in the cluster (Scha ¨pe et al. 2019). It
may therefore be desirable to reconstruct the
genes involved in formation of the heterologous
compound using a setup that allows necessary
GOIs to be equipped with known and wellcharacterized promoter and terminator
sequences. Here we present three strategies
that allow for bio-block-based multi-GOI
expression (Fig. 10.3). Although multi-GOI
expression cassettes can be assembled on
AMA1-based vectors, we recommend using
defined chromosomal expression sites for GOI
insertion to gain genetic stability and to reduce
dependency on selectable markers.
In the first strategy (Fig. 10.3a), individual
expression cassettes for different GOIs are
inserted into a number of unique integration
sites. This strategy is advisable if all of the
individual gene-expression cassettes contain
an identical bio-block, such as a specific promoter or terminator. In this way, copy loss due
to direct repeat recombination between the
identical bio-blocks is avoided. With conventional gene-targeting methods, insertion of
gene-expression cassettes into individual sites
requires a number of selectable markers that
matches the number of gene-expression cassettes required for establishing the biosynthetic
pathway. Alternatively, the genes can be
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
231
comparative screening of expression levels or
enzyme activities based on different genetic
elements employed in the cassette, such as promoter, secretion signal, and terminator
sequences. Similarly, the effects achieved by
changing the GOI sequence can be directly
compared. In this way it is possible to address
whether changes in the GOI codon composition
increase yields or whether mutations infer
changes in the heterologous protein that influences its activity, specificity, folding, and/or
stability (Hansen et al. 2011b; Holm 2013).
Gene-expression cassettes can be inserted
into integration sites in the genome by HR. To
ensure high expression levels, it may be useful to
position integration sites in intergenic regions
located in transcriptionally highly active sections of a chromosome. With this method, the
gene-expression cassettes must be flanked by
up- and downstream targeting sequences matching the genomic expression site. Since fungi
rarely prefer to integrate foreign DNA into its
genome by the HR pathway, extensive screening
for the desired transformant may be necessary.
The screening workload can be reduced by using
bipartite gene-targeting substrates that select
for HR proficient protoplasts (Nielsen et al.
2006) or avoided by using NHEJ-deficient
strains (Meyer et al. 2007). Alternatively, methods based on restriction enzymes or CRISPR
technology can be employed (Ouedraogo et al.
2016; Zheng et al. 2017). Importantly with the
latter technology, GOIs can be inserted in a
marker-free manner and multiplexing is possible (Liu et al. 2015; Nødvig et al. 2018; Zhang
et al. 2016a). In fact, CRISPR mediates very
efficient gene targeting even in NHEJ-proficient
strains (Nødvig et al. 2018), eliminating the risks
of working with strains with a defective DNA
repair pathway. We therefore envision that
CRISPR-based methods will deliver the preferred tools for introducing gene-expression cassettes into defined chromosomal expression
platforms.
C. Bio-Block-Based Multi-GOI Expression
Strategies
In many cases, two or more genes are required
for the synthesis of a desired product. For
example, for heterologous production of SMs,
several genes are commonly required to synthesize the product. Above, we described that it is
possible to transfer large chromosomal fragments containing entire gene clusters from
one fungus to another (Sect. II.B.1). However,
in many cases the activation mechanism for
the genes in the cluster is not known and
may depend on unknown or even host-specific
transcription factors (TF) (Keller 2019); see
Chap. 11. Moreover, not all genes contributing
to the biosynthetic pathway of interest may be
situated in the cluster (Scha ¨pe et al. 2019). It
may therefore be desirable to reconstruct the
genes involved in formation of the heterologous
compound using a setup that allows necessary
GOIs to be equipped with known and wellcharacterized promoter and terminator
sequences. Here we present three strategies
that allow for bio-block-based multi-GOI
expression (Fig. 10.3). Although multi-GOI
expression cassettes can be assembled on
AMA1-based vectors, we recommend using
defined chromosomal expression sites for GOI
insertion to gain genetic stability and to reduce
dependency on selectable markers.
In the first strategy (Fig. 10.3a), individual
expression cassettes for different GOIs are
inserted into a number of unique integration
sites. This strategy is advisable if all of the
individual gene-expression cassettes contain
an identical bio-block, such as a specific promoter or terminator. In this way, copy loss due
to direct repeat recombination between the
identical bio-blocks is avoided. With conventional gene-targeting methods, insertion of
gene-expression cassettes into individual sites
requires a number of selectable markers that
matches the number of gene-expression cassettes required for establishing the biosynthetic
pathway. Alternatively, the genes can be
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
231