used markers are niaD required for nitrate
assimilation (Unkles et al. 1989) and the amdS
gene from A. nidulans allowing growth on acetamide as the sole nitrogen source (Kelly and
Hynes 1985).
Like visual markers, the design of the nutritional marker can be used to select for strains
containing multiple expression cassettes. For
example, the complementary marker gene can
be equipped with a poor promoter to ensure
that only strains with many copies produce
sufficient amounts of the missing enzyme.
Often several auxotrophies are introduced
in the host to allow for several rounds of genetic
engineering using a set of complementing
nutritional markers. It is important to note
that nutritional markers may change the
metabolism of the host cell despite that the
defects are compensated by addition of the
missing metabolites to the growth medium.
For physiological characterizations of the engineered strains, this may lead to undesired artefacts; and for cell factories, it may lead to
suboptimal growth, or growth, which is
restricted to specific media. It is therefore
advisable to restore the functionality of those
nutritional markers after genetic engineering.
To facilitate this process, it has been recently
shown that it is possible to functionally revert
several mutated genes by multiplexing CRISPR
technology (Nødvig et al. 2018).
4. Counter-Selectable Markers
Some nutritional markers can be counterselected by using analogs of their natural substrates that are converted into antimetabolites.
The two most frequently used counterselectable wild-type markers are pyrG and
amdS, which are lethal in the presence of 5fluoroorotic acid (5-FOA) and 5
0 -fluoroacetamide (FAA), respectively. For both pyrG, and
to lesser extent amdS, a gene copy is naturally
present in wild-type fungi. Hence, the endogenous gene needs to be disrupted before the
marker can be applied in selection/counterselection experiments. In pyrG
+ strains, 5-FOA
is converted by the orotidine-5
0 -phosphate
decarboxylase (PyrG) into 5-fluorouracil,
which is further metabolized into toxic substances that interfere with RNA and DNA synthesis (Boeke et al. 1984; Longley et al. 2003).
Similarly, in amdS
+ strains, FAA is converted
by the acetamidase (AmdS) into the toxic compound fluoroacetate (Hynes and Pateman
1970), which forms a stable complex with Coenzyme A (fluoroacetyl-CoA), hence hindering
normal functionality of the tricarboxylic acid
cycle. Other counter-selectable markers include
genes in the tryptophan pathway, most often
trpA (Foureau et al. 2012), and genes in the
lysine pathway, most often lysB (Alberti et al.
2003), which can be counter-selected in the
presence of 5-fluoroanthranilic acid, 5-FAA,
and a-aminoadipate, respectively. Similarly,
expression of genes encoding herpes simplex
virus 1 thymidine kinase can be counterselected as the viral thymidine kinase, unlike
the thymidine kinases of the hosts, activates
nucleoside analogs like 5-fluoro-2
0 -deoxyuridine for toxic incorporation into nucleic acids
(Lupton et al. 1991). Counter-selectable markers are highly useful as they facilitate marker
recycling and iterative genetic engineering.
5. Marker Recycling for Iterative Engineering
In most fungi, the number of available markers
is a limiting factor for multi-step genetic engineering strategies. If, e.g., an entire biosynthetic
pathway needs to be implemented into a new
host, marker recycling is therefore often a
necessity. Specifically, if two markers are available for transformation of a host strain, an
endless number of genes can in principle be
inserted into the same site in the host genome
by iterative marker-swapping. In this method,
the marker gene, which was used for gene integration in the previous transformation step, is
replaced by a new gene targeting construct that
contains new GOIs and another marker (Nielsen et al. 2013).
A marker gene can also be eliminated from
the genome by spontaneous direct-repeat
recombination if the marker is flanked by
sufficiently long direct repeats, typically
500–1000 bp (Nielsen et al. 2006). This method
may be preferred if it is desirable to leave a
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
237
assimilation (Unkles et al. 1989) and the amdS
gene from A. nidulans allowing growth on acetamide as the sole nitrogen source (Kelly and
Hynes 1985).
Like visual markers, the design of the nutritional marker can be used to select for strains
containing multiple expression cassettes. For
example, the complementary marker gene can
be equipped with a poor promoter to ensure
that only strains with many copies produce
sufficient amounts of the missing enzyme.
Often several auxotrophies are introduced
in the host to allow for several rounds of genetic
engineering using a set of complementing
nutritional markers. It is important to note
that nutritional markers may change the
metabolism of the host cell despite that the
defects are compensated by addition of the
missing metabolites to the growth medium.
For physiological characterizations of the engineered strains, this may lead to undesired artefacts; and for cell factories, it may lead to
suboptimal growth, or growth, which is
restricted to specific media. It is therefore
advisable to restore the functionality of those
nutritional markers after genetic engineering.
To facilitate this process, it has been recently
shown that it is possible to functionally revert
several mutated genes by multiplexing CRISPR
technology (Nødvig et al. 2018).
4. Counter-Selectable Markers
Some nutritional markers can be counterselected by using analogs of their natural substrates that are converted into antimetabolites.
The two most frequently used counterselectable wild-type markers are pyrG and
amdS, which are lethal in the presence of 5fluoroorotic acid (5-FOA) and 5
0 -fluoroacetamide (FAA), respectively. For both pyrG, and
to lesser extent amdS, a gene copy is naturally
present in wild-type fungi. Hence, the endogenous gene needs to be disrupted before the
marker can be applied in selection/counterselection experiments. In pyrG
+ strains, 5-FOA
is converted by the orotidine-5
0 -phosphate
decarboxylase (PyrG) into 5-fluorouracil,
which is further metabolized into toxic substances that interfere with RNA and DNA synthesis (Boeke et al. 1984; Longley et al. 2003).
Similarly, in amdS
+ strains, FAA is converted
by the acetamidase (AmdS) into the toxic compound fluoroacetate (Hynes and Pateman
1970), which forms a stable complex with Coenzyme A (fluoroacetyl-CoA), hence hindering
normal functionality of the tricarboxylic acid
cycle. Other counter-selectable markers include
genes in the tryptophan pathway, most often
trpA (Foureau et al. 2012), and genes in the
lysine pathway, most often lysB (Alberti et al.
2003), which can be counter-selected in the
presence of 5-fluoroanthranilic acid, 5-FAA,
and a-aminoadipate, respectively. Similarly,
expression of genes encoding herpes simplex
virus 1 thymidine kinase can be counterselected as the viral thymidine kinase, unlike
the thymidine kinases of the hosts, activates
nucleoside analogs like 5-fluoro-2
0 -deoxyuridine for toxic incorporation into nucleic acids
(Lupton et al. 1991). Counter-selectable markers are highly useful as they facilitate marker
recycling and iterative genetic engineering.
5. Marker Recycling for Iterative Engineering
In most fungi, the number of available markers
is a limiting factor for multi-step genetic engineering strategies. If, e.g., an entire biosynthetic
pathway needs to be implemented into a new
host, marker recycling is therefore often a
necessity. Specifically, if two markers are available for transformation of a host strain, an
endless number of genes can in principle be
inserted into the same site in the host genome
by iterative marker-swapping. In this method,
the marker gene, which was used for gene integration in the previous transformation step, is
replaced by a new gene targeting construct that
contains new GOIs and another marker (Nielsen et al. 2013).
A marker gene can also be eliminated from
the genome by spontaneous direct-repeat
recombination if the marker is flanked by
sufficiently long direct repeats, typically
500–1000 bp (Nielsen et al. 2006). This method
may be preferred if it is desirable to leave a
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
237