transformation in A. nidulans (Johnstone et al.
1985). In subsequent studies, it has been shown
that AMA1-based vectors increase fungal transformation efficiency in A. nidulans up to 2000
times in comparison to earlier developed ectopically integrating vectors (Aleksenko and Clutterbuck 1997). Moreover, AMA1 plasmids
propagate in other Aspergillus species as well
as in species belonging to other fungal genera,
including Penicillium (Fierro et al. 1996),
Talaromyces (Nielsen et al. 2017), Trichoderma
(Kubodera et al. 2002), and Rosellinia (Shimizu
et al. 2012).
Gene-expression cassettes can easily be
incorporated into extrachromosomal AMA1based shuttle vectors, using the cloning systems highlighted above. As an extreme case,
fungal AMA1 plasmids have been combined
with a bacterial artificial chromosome (Zhu
et al. 1997) to form fungal artificial chromosomes (FACs). These vectors have been instrumental in the search for new secondary
metabolite gene clusters as they allow up to
300 kbp of fungal DNA to be cloned into the
FAC in E. coli and subsequently transferred into
a new fungal host for product discovery (Bok
et al. 2015; Clevenger et al. 2017). Importantly,
AMA1-based plasmids are not very stable and
their maintenance requires selection, thereby
limiting their use to small-scale exploratory
heterologous production experiments. However, we note that for some purposes, such as
cas9 expression in CRISPR experiments, plasmid instability is desirable as plasmid loss
allows for transient gene expression, diminishing potential Cas9 off-target effects (Zhang et al.
2015).
2. Chromosome-Based Expression Systems
Integration of the expression cassette into the
host genome provides a more stable expression,
as compared to AMA1-based expression, and
attenuates the need to maintain selection pressure. Chromosomal integration of foreign DNA
occurs via one of the two DNA repair mechanisms (Krappmann 2007), either non-homologous end joining (NHEJ), where the DNA
integrates into a random locus (Fig. 10.2b), or
homologous recombination (HR), where the
DNA integrates into a defined locus
(Fig. 10.2c). Although both pathways are active
in all fungi, foreign DNA typically integrates
more efficiently via the NHEJ pathway in most
filamentous fungal species (Meyer et al. 2007;
Nødvig et al. 2015).
a) Random Chromosomal Integration of GeneExpression Cassettes
Since NHEJ is the dominant pathway for chromosomal integration of DNA in most filamentous fungi, random chromosomal integration
of gene-expression cassettes via NHEJ is therefore straightforward and can be efficiently performed in essentially all transformable fungal
species. A drawback of the method is that the
chromatin structures of the insertion sites are
unpredictable and expression levels may therefore vary substantially between different transformants (Lubertozzi and Keasling 2006). On
the other hand, if several gene-expression cassettes enter a nucleus, they may undergo
recombination prior to genomic integration.
As a result, transformants may contain multiple
copies of the GOI inserted into a single locus. In
these cases, the transformants will often be better producers of the product due to the many
gene copies. However, since the cassettes will be
organized as direct and/or inverted repeats,
expression levels of such strains may be unstable as gene copies may be lost due to direct
repeat recombination or formation of hairpin
structures (Leach 1994; Petes 1988). Lastly, it is
important to note that when gene-expression
cassettes integrate randomly into the genome,
they may disrupt important genes, or alter the
expression levels of neighboring genes, causing
undesired phenotypes. In summary, with this
integration method, it is advisable to screen for
transformants that produce high yields over
many generations and at the same time do not
display undesirable fitness defects.
b) Defined Chromosomal Integration of GeneExpression Cassettes
The use of defined and well-characterized loci
as integration sites for GECs allows for gene
expression that is much less prone to clonal
230
J. K. H. Rendsvig et al.
1985). In subsequent studies, it has been shown
that AMA1-based vectors increase fungal transformation efficiency in A. nidulans up to 2000
times in comparison to earlier developed ectopically integrating vectors (Aleksenko and Clutterbuck 1997). Moreover, AMA1 plasmids
propagate in other Aspergillus species as well
as in species belonging to other fungal genera,
including Penicillium (Fierro et al. 1996),
Talaromyces (Nielsen et al. 2017), Trichoderma
(Kubodera et al. 2002), and Rosellinia (Shimizu
et al. 2012).
Gene-expression cassettes can easily be
incorporated into extrachromosomal AMA1based shuttle vectors, using the cloning systems highlighted above. As an extreme case,
fungal AMA1 plasmids have been combined
with a bacterial artificial chromosome (Zhu
et al. 1997) to form fungal artificial chromosomes (FACs). These vectors have been instrumental in the search for new secondary
metabolite gene clusters as they allow up to
300 kbp of fungal DNA to be cloned into the
FAC in E. coli and subsequently transferred into
a new fungal host for product discovery (Bok
et al. 2015; Clevenger et al. 2017). Importantly,
AMA1-based plasmids are not very stable and
their maintenance requires selection, thereby
limiting their use to small-scale exploratory
heterologous production experiments. However, we note that for some purposes, such as
cas9 expression in CRISPR experiments, plasmid instability is desirable as plasmid loss
allows for transient gene expression, diminishing potential Cas9 off-target effects (Zhang et al.
2015).
2. Chromosome-Based Expression Systems
Integration of the expression cassette into the
host genome provides a more stable expression,
as compared to AMA1-based expression, and
attenuates the need to maintain selection pressure. Chromosomal integration of foreign DNA
occurs via one of the two DNA repair mechanisms (Krappmann 2007), either non-homologous end joining (NHEJ), where the DNA
integrates into a random locus (Fig. 10.2b), or
homologous recombination (HR), where the
DNA integrates into a defined locus
(Fig. 10.2c). Although both pathways are active
in all fungi, foreign DNA typically integrates
more efficiently via the NHEJ pathway in most
filamentous fungal species (Meyer et al. 2007;
Nødvig et al. 2015).
a) Random Chromosomal Integration of GeneExpression Cassettes
Since NHEJ is the dominant pathway for chromosomal integration of DNA in most filamentous fungi, random chromosomal integration
of gene-expression cassettes via NHEJ is therefore straightforward and can be efficiently performed in essentially all transformable fungal
species. A drawback of the method is that the
chromatin structures of the insertion sites are
unpredictable and expression levels may therefore vary substantially between different transformants (Lubertozzi and Keasling 2006). On
the other hand, if several gene-expression cassettes enter a nucleus, they may undergo
recombination prior to genomic integration.
As a result, transformants may contain multiple
copies of the GOI inserted into a single locus. In
these cases, the transformants will often be better producers of the product due to the many
gene copies. However, since the cassettes will be
organized as direct and/or inverted repeats,
expression levels of such strains may be unstable as gene copies may be lost due to direct
repeat recombination or formation of hairpin
structures (Leach 1994; Petes 1988). Lastly, it is
important to note that when gene-expression
cassettes integrate randomly into the genome,
they may disrupt important genes, or alter the
expression levels of neighboring genes, causing
undesired phenotypes. In summary, with this
integration method, it is advisable to screen for
transformants that produce high yields over
many generations and at the same time do not
display undesirable fitness defects.
b) Defined Chromosomal Integration of GeneExpression Cassettes
The use of defined and well-characterized loci
as integration sites for GECs allows for gene
expression that is much less prone to clonal
230
J. K. H. Rendsvig et al.