genetically neutral DNA sequence (i.e. the
direct repeat) as the only scar after genetic
engineering, or if it is desirable to integrate
several genes or sets of genes into several different loci in the genome. As spontaneous
direct recombination events are rare, this
method requires that marker loss is selectable,
e.g., by using a counter-selectable marker; see
above (Sect. III.B.4). Alternatively, marker loss
can be achieved by induced recombination
involving either site-specific recombinases like
Cre and a marker, which is flanked by its target
sequences, or gene deletion catalyzed by sitespecific nucleases like I-SceI or a CRISPR
nuclease (Ouedraogo et al. 2016; Zhang et al.
2013).
C. Promoters
Among the different bio-blocks, promoters
have drawn most attention since they control
gene transcription initiation. Promoters are
typically divided in two groups: those that are
constitutively active, i.e., promoters that are
active under all/most circumstances, and those
that are inducible/repressible. Often constitutive promoters are preferred for large-scale heterologous production as they are active
throughout the fermentation process in inexpensive media. In cases where the product is
toxic or unstable, it may be necessary to restrict
production to a specific growth phase to maximize yields; and in these cases, it is necessary to
use an inducible/repressible promoter. However, as addition of inducing or repressing
agents comes with an additional cost, use of
this type of promoters may be restricted to
small scale production or exploratory studies.
The vast majority of heterologous geneexpression studies are based on natural promoters of which we have listed sets of frequently
used promoters derived from different fungal
genera (Table 10.2). Interestingly, many promoters involved in basic metabolism do not
seem to be species specific. Hence, promoter
versatility allows the same genetic element to
be used in many different species, even species
belonging to different genera. However, to our
knowledge, no comparative studies have systematically analyzed how active a given promoter is in different species. Based on the fair
assumption that a promoter is most active in
the species from which it originates, and less
active when applied in other fungi, it is advisable to equip the GOI with a promoter derived
from the intended production host. Importantly, the efficiency of promoters, even constitutive promoters, often depends on the growth
environment and growth phase of the host. This
has motivated establishment of synthetic promoters that offer orthogonal setups that work
independently of the host metabolism. Below
we present examples of natural and synthetic
promoters.
1. Natural Promoters
A wide selection of natural fungal promoters
for species belonging to Aspergillus, Penicillium, and Trichoderma genera as well as others
(Table 10.2) have been experimentally characterized as constitutive or inducible, along with
their mode of induction and repression, as
reviewed in Fitz et al. (2018), Fleissner and
Dersch (2010), and Kluge et al. (2018). Typical
constitutive promoters for heterologous
expression in fungal cell factories are derived
from genes involved in major metabolic pathways or cell functions that require high steady
protein levels. Examples are the gpdA promoter
(PgpdA) and the stronger tef1 promoter (Ptef1),
which control production of glyceraldehyde-3phosphate dehydrogenase acting in glycolysis
and translation elongation factor 1a assisting in
protein synthesis, respectively. Promoters catalyzing basic reactions in the cell are often functional if transferred to other species. In line
with this view, PgpdA and Ptef1 from A. nidulans have been shown to work in other species
of Aspergilli (Nødvig et al. 2015), in Talaromyces atroroseus (Nielsen et al. 2017), and the
PgpdA of A. nidulans was even applied in the
fungus Metarhizium anisopliae (Nakazato et al.
2006). Inducible/repressible promoters are
also recruited from basic metabolic genes.
This includes the commonly used alcohol and
threonine inducible alcA promoter, which controls expression of alcohol dehydrogenase I
238
J. K. H. Rendsvig et al.
direct repeat) as the only scar after genetic
engineering, or if it is desirable to integrate
several genes or sets of genes into several different loci in the genome. As spontaneous
direct recombination events are rare, this
method requires that marker loss is selectable,
e.g., by using a counter-selectable marker; see
above (Sect. III.B.4). Alternatively, marker loss
can be achieved by induced recombination
involving either site-specific recombinases like
Cre and a marker, which is flanked by its target
sequences, or gene deletion catalyzed by sitespecific nucleases like I-SceI or a CRISPR
nuclease (Ouedraogo et al. 2016; Zhang et al.
2013).
C. Promoters
Among the different bio-blocks, promoters
have drawn most attention since they control
gene transcription initiation. Promoters are
typically divided in two groups: those that are
constitutively active, i.e., promoters that are
active under all/most circumstances, and those
that are inducible/repressible. Often constitutive promoters are preferred for large-scale heterologous production as they are active
throughout the fermentation process in inexpensive media. In cases where the product is
toxic or unstable, it may be necessary to restrict
production to a specific growth phase to maximize yields; and in these cases, it is necessary to
use an inducible/repressible promoter. However, as addition of inducing or repressing
agents comes with an additional cost, use of
this type of promoters may be restricted to
small scale production or exploratory studies.
The vast majority of heterologous geneexpression studies are based on natural promoters of which we have listed sets of frequently
used promoters derived from different fungal
genera (Table 10.2). Interestingly, many promoters involved in basic metabolism do not
seem to be species specific. Hence, promoter
versatility allows the same genetic element to
be used in many different species, even species
belonging to different genera. However, to our
knowledge, no comparative studies have systematically analyzed how active a given promoter is in different species. Based on the fair
assumption that a promoter is most active in
the species from which it originates, and less
active when applied in other fungi, it is advisable to equip the GOI with a promoter derived
from the intended production host. Importantly, the efficiency of promoters, even constitutive promoters, often depends on the growth
environment and growth phase of the host. This
has motivated establishment of synthetic promoters that offer orthogonal setups that work
independently of the host metabolism. Below
we present examples of natural and synthetic
promoters.
1. Natural Promoters
A wide selection of natural fungal promoters
for species belonging to Aspergillus, Penicillium, and Trichoderma genera as well as others
(Table 10.2) have been experimentally characterized as constitutive or inducible, along with
their mode of induction and repression, as
reviewed in Fitz et al. (2018), Fleissner and
Dersch (2010), and Kluge et al. (2018). Typical
constitutive promoters for heterologous
expression in fungal cell factories are derived
from genes involved in major metabolic pathways or cell functions that require high steady
protein levels. Examples are the gpdA promoter
(PgpdA) and the stronger tef1 promoter (Ptef1),
which control production of glyceraldehyde-3phosphate dehydrogenase acting in glycolysis
and translation elongation factor 1a assisting in
protein synthesis, respectively. Promoters catalyzing basic reactions in the cell are often functional if transferred to other species. In line
with this view, PgpdA and Ptef1 from A. nidulans have been shown to work in other species
of Aspergilli (Nødvig et al. 2015), in Talaromyces atroroseus (Nielsen et al. 2017), and the
PgpdA of A. nidulans was even applied in the
fungus Metarhizium anisopliae (Nakazato et al.
2006). Inducible/repressible promoters are
also recruited from basic metabolic genes.
This includes the commonly used alcohol and
threonine inducible alcA promoter, which controls expression of alcohol dehydrogenase I
238
J. K. H. Rendsvig et al.