antimicrobial markers, visual markers can be
used for selection in wild-type strains, but without posing any risks in relation to compound
toxicity or transference of resistance genes to
other organisms.
Three types of heterologous visual markers are frequently used, all of which may be
applied for spectrophotometric assays. Firstly,
b-galactosidase LacZ (lacZ) (Lubertozzi and
Keasling 2006) and b-glucuronidase GusA
(uidA) of E. coli (Tada et al. 1991), respectively,
convert the synthetic substrates X-gal and Xgluc to 5-bromo-4-chloro-3-hydroxyindole that
undergoes dimerization forming a blue pigment. Secondly, luciferases convert substrates
into bioluminescent products. Thirdly, fluorescent proteins derived from jellyfish Aequorea
victoria GFP, which was adapted for fungal use
by codon optimization and by eliminating a
non-conventional/cryptic intron splice site
(Lorang et al. 2001), or from RFP.
Visual markers do not offer any fitness
advantage, and, if the goal is to detect transformants containing the expression cassette, selection requires visual screening. Selection by
solely visual inspection can be achieved by
inserting the cassette into conidial pigmentation genes (e.g., homologs of A. nidulans wA
and yA genes), resulting in a distinct change in
conidial pigmentation from the native black or
green spores of Aspergillus species to white or
yellow (Jørgensen et al. 2011; Nielsen et al.
2006). Similarly, the expression cassette can be
inserted into adeA or adeB as this will produce
easy detectable red colonies due to polymerization and oxidation of 4-amino-imidazole ribotide, the intermediate resulting from the
disrupted purine biosynthetic pathway (Jin
et al. 2004). Alternatively, the gene-expression
cassette can be equipped with a heterologous
marker that produces color, fluorescence, or
bioluminescence to allow for visual selection.
This is useful if the goal is to insert the cassette
into an intergenic section of the genome by HR,
thereby avoiding host gene disruption. In cases
where correct transformants are rare, fluorescent markers set the stage for high-throughput
detection schemes via FACS analysis, which
can be employed if the fungus produces discrete entities like conidia (Bleichrodt and Read
2019; Vlaardingerbroek et al. 2015). Color, fluorescent, or bioluminescent markers can also be
advantageously used if the goal is to insert the
gene-expression cassette in multiple copies,
e.g., via integration events catalyzed by the
NHEJ pathway, as transformants with a high
copy number can be selected for by the strength
of the marker signal (Throndset et al. 2010).
When heterologous enzymes and fluorescent proteins are used as selective markers, it
should be noted that some fungi produce
enzymes that may catalyze the same reactions
as those provided by the marker enzyme, or the
fungi may display auto-fluorescence. It is therefore always necessary to test whether significant
native background signals exist.
3. Nutritional Markers
Usage of nutritional markers relay on auxotrophies in the host organism, which thereby
requires supplementation with specific metabolites to sustain growth. Introducing a functional copy of the given nutritional marker into
the host complements the defective native gene
function, allowing for growth without supplementation. In this way, nutritional markers
offer selective pressure without the negative
effects of antibiotics. On the other hand, prior
to cell factory construction, the host needs to be
mutagenized or genetically engineered to create
the relevant auxotrophic mutations. If the
starting point is a wild-type strain, this may
require the use of mutagens, traditional genetic
engineering using antimicrobial or visual markers, or CRISPR technology. Complementing
homologous or heterologous genes can be
used as markers, but often a heterologous
marker is preferred as the sequence differences
reduce the risk of generating false positives due
to homologous recombination between the
marker and the corresponding mutated locus.
Some commonly used nutritional markers
include argB (Buxton et al. 1985), trpC (Goosen
et al. 1989), adeA and adeB (Jin et al. 2004),
pyroA (Osmani et al. 1999), and pyrG (Goosen
et al. 1987), which are required for synthesis of
arginine, tryptophan, adenine, pyridoxine, and
uracil/uridine, respectively. Other frequently
236
J. K. H. Rendsvig et al.
used for selection in wild-type strains, but without posing any risks in relation to compound
toxicity or transference of resistance genes to
other organisms.
Three types of heterologous visual markers are frequently used, all of which may be
applied for spectrophotometric assays. Firstly,
b-galactosidase LacZ (lacZ) (Lubertozzi and
Keasling 2006) and b-glucuronidase GusA
(uidA) of E. coli (Tada et al. 1991), respectively,
convert the synthetic substrates X-gal and Xgluc to 5-bromo-4-chloro-3-hydroxyindole that
undergoes dimerization forming a blue pigment. Secondly, luciferases convert substrates
into bioluminescent products. Thirdly, fluorescent proteins derived from jellyfish Aequorea
victoria GFP, which was adapted for fungal use
by codon optimization and by eliminating a
non-conventional/cryptic intron splice site
(Lorang et al. 2001), or from RFP.
Visual markers do not offer any fitness
advantage, and, if the goal is to detect transformants containing the expression cassette, selection requires visual screening. Selection by
solely visual inspection can be achieved by
inserting the cassette into conidial pigmentation genes (e.g., homologs of A. nidulans wA
and yA genes), resulting in a distinct change in
conidial pigmentation from the native black or
green spores of Aspergillus species to white or
yellow (Jørgensen et al. 2011; Nielsen et al.
2006). Similarly, the expression cassette can be
inserted into adeA or adeB as this will produce
easy detectable red colonies due to polymerization and oxidation of 4-amino-imidazole ribotide, the intermediate resulting from the
disrupted purine biosynthetic pathway (Jin
et al. 2004). Alternatively, the gene-expression
cassette can be equipped with a heterologous
marker that produces color, fluorescence, or
bioluminescence to allow for visual selection.
This is useful if the goal is to insert the cassette
into an intergenic section of the genome by HR,
thereby avoiding host gene disruption. In cases
where correct transformants are rare, fluorescent markers set the stage for high-throughput
detection schemes via FACS analysis, which
can be employed if the fungus produces discrete entities like conidia (Bleichrodt and Read
2019; Vlaardingerbroek et al. 2015). Color, fluorescent, or bioluminescent markers can also be
advantageously used if the goal is to insert the
gene-expression cassette in multiple copies,
e.g., via integration events catalyzed by the
NHEJ pathway, as transformants with a high
copy number can be selected for by the strength
of the marker signal (Throndset et al. 2010).
When heterologous enzymes and fluorescent proteins are used as selective markers, it
should be noted that some fungi produce
enzymes that may catalyze the same reactions
as those provided by the marker enzyme, or the
fungi may display auto-fluorescence. It is therefore always necessary to test whether significant
native background signals exist.
3. Nutritional Markers
Usage of nutritional markers relay on auxotrophies in the host organism, which thereby
requires supplementation with specific metabolites to sustain growth. Introducing a functional copy of the given nutritional marker into
the host complements the defective native gene
function, allowing for growth without supplementation. In this way, nutritional markers
offer selective pressure without the negative
effects of antibiotics. On the other hand, prior
to cell factory construction, the host needs to be
mutagenized or genetically engineered to create
the relevant auxotrophic mutations. If the
starting point is a wild-type strain, this may
require the use of mutagens, traditional genetic
engineering using antimicrobial or visual markers, or CRISPR technology. Complementing
homologous or heterologous genes can be
used as markers, but often a heterologous
marker is preferred as the sequence differences
reduce the risk of generating false positives due
to homologous recombination between the
marker and the corresponding mutated locus.
Some commonly used nutritional markers
include argB (Buxton et al. 1985), trpC (Goosen
et al. 1989), adeA and adeB (Jin et al. 2004),
pyroA (Osmani et al. 1999), and pyrG (Goosen
et al. 1987), which are required for synthesis of
arginine, tryptophan, adenine, pyridoxine, and
uracil/uridine, respectively. Other frequently
236
J. K. H. Rendsvig et al.