2. Genome Annotation and Intron Splicing
Heterologous production requires that the
genes in the native producers are correctly
annotated and for many sequenced genes, this
may not be the case. In cases of doubt, it may be
necessary to determine the correct 5
0
-end of the
transcript by the rapid amplification of cDNA
ends (RACE) technique (Frohman et al. 1988).
Subsequently, introns and the correct stop
codon can be identified by generating a complete cDNA of the gene by RT-PCR exploiting
that the 3
0 -end of the transcript is polyadenylated. However, in cases where the GOI is silent,
this strategy is not possible, and it may therefore be advisable to produce a set of gene constructs covering different combinations of start
and stop codon possibilities.
Intron recognition and splicing differs
between species, especially for phylogenetically
distant organisms (Kupfer et al. 2004), and
flawed splicing may reduce or even prevent
formation of the desired protein as described
in Sect. III.A.1. For example, introns are more
abundant in basidiomycetes than in ascomycetes (Stajich et al. 2007). Moreover, introns
from basidiomycetes may not be recognized
properly in ascomycetes.
In agreement with this, a recent study using A. oryzae
as a cell factory demonstrated that only half of the
mRNA species produced by 30 terpene synthase genes
from two basidiomycetes, Clitopilus pseudo-pinsitus
and Stereum hirsutum, exhibited correct splicing patterns (Nagamine et al. 2019).
Even among species that are more closely
related, e.g., within ascomycetes, transfer of
genes between species may cause splicing
errors. For example, heterologous expression
of the 3-methylorcinaldehyde synthase gene
from Acremonium strictum (Fisch et al. 2010),
the avirulence gene ACE1 from Magnaporthe
oryzae (Song et al. 2015), and the citrinin
synthase gene from Monascus ruber (He and
Cox 2016) in A. oryzae resulted in incorrect
processing of introns.
Moreover, some mRNAs of filamentous
fungi are regulated by alternative splicing
(Kempken 2013; Zhao et al. 2013). Hence,
populations of different splice variants were
detected in the transcriptome (Wang et al.
2010) with consequences for protein levels
(Chang et al. 2010). Indeed, new splice variants
may be inactive or the correct variant may be
produced only in small amounts (He and Cox
2016; Kempken and Windhofer 2004). Mapping
mRNA splicing in the native producer, if possible, is therefore advisable; and if it is different
in the new host, it may be necessary to produce
different synthetic gene variants covering different splice variants to achieve heterologous
production.
3. Compartmentalization of Secondary
Metabolite Biosynthetic Pathways
Fungal secondary metabolite biosynthetic pathways are often compartmentalized into different subcellular locations, such as ER, vesicles,
peroxisomes, cytoplasm, or vacuoles (Kistler
and Broz 2015; Roze et al. 2011). The reason
for this is either to channel the pathway into
the compartment with relevant precursors to
increase biosynthesis efficiency as in the case
of penicillin in A. nidulans (Herr and Fischer
2014) or to sequester the toxic product or
intermediates from the rest of the cell as in the
case of aflatoxin synthesis in Aspergillus parasiticus (Chanda et al. 2009). Little is known
about how SM biosynthetic pathways are compartmentalized in fungi, and it is unclear how
often this occurs. However, in cases where compartmentalization is important for product formation or to avoid toxicity effects, it is likely
essential that the pathway is organized in a
similar manner in a new host. Otherwise, it
may result in lack of production or even cell
death. In the future, we expect that pathway
compartmentalization will achieve significant
attention in SM cell factory construction. As a
standard control experiment, we advise to
determine whether individual enzymes in the
pathway localize to the same compartments in
the natural producer and in the new host, e.g.,
by GFP tagging. In cases where the host sorts an
enzyme incorrectly, it may be possible to
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
251
Heterologous production requires that the
genes in the native producers are correctly
annotated and for many sequenced genes, this
may not be the case. In cases of doubt, it may be
necessary to determine the correct 5
0
-end of the
transcript by the rapid amplification of cDNA
ends (RACE) technique (Frohman et al. 1988).
Subsequently, introns and the correct stop
codon can be identified by generating a complete cDNA of the gene by RT-PCR exploiting
that the 3
0 -end of the transcript is polyadenylated. However, in cases where the GOI is silent,
this strategy is not possible, and it may therefore be advisable to produce a set of gene constructs covering different combinations of start
and stop codon possibilities.
Intron recognition and splicing differs
between species, especially for phylogenetically
distant organisms (Kupfer et al. 2004), and
flawed splicing may reduce or even prevent
formation of the desired protein as described
in Sect. III.A.1. For example, introns are more
abundant in basidiomycetes than in ascomycetes (Stajich et al. 2007). Moreover, introns
from basidiomycetes may not be recognized
properly in ascomycetes.
In agreement with this, a recent study using A. oryzae
as a cell factory demonstrated that only half of the
mRNA species produced by 30 terpene synthase genes
from two basidiomycetes, Clitopilus pseudo-pinsitus
and Stereum hirsutum, exhibited correct splicing patterns (Nagamine et al. 2019).
Even among species that are more closely
related, e.g., within ascomycetes, transfer of
genes between species may cause splicing
errors. For example, heterologous expression
of the 3-methylorcinaldehyde synthase gene
from Acremonium strictum (Fisch et al. 2010),
the avirulence gene ACE1 from Magnaporthe
oryzae (Song et al. 2015), and the citrinin
synthase gene from Monascus ruber (He and
Cox 2016) in A. oryzae resulted in incorrect
processing of introns.
Moreover, some mRNAs of filamentous
fungi are regulated by alternative splicing
(Kempken 2013; Zhao et al. 2013). Hence,
populations of different splice variants were
detected in the transcriptome (Wang et al.
2010) with consequences for protein levels
(Chang et al. 2010). Indeed, new splice variants
may be inactive or the correct variant may be
produced only in small amounts (He and Cox
2016; Kempken and Windhofer 2004). Mapping
mRNA splicing in the native producer, if possible, is therefore advisable; and if it is different
in the new host, it may be necessary to produce
different synthetic gene variants covering different splice variants to achieve heterologous
production.
3. Compartmentalization of Secondary
Metabolite Biosynthetic Pathways
Fungal secondary metabolite biosynthetic pathways are often compartmentalized into different subcellular locations, such as ER, vesicles,
peroxisomes, cytoplasm, or vacuoles (Kistler
and Broz 2015; Roze et al. 2011). The reason
for this is either to channel the pathway into
the compartment with relevant precursors to
increase biosynthesis efficiency as in the case
of penicillin in A. nidulans (Herr and Fischer
2014) or to sequester the toxic product or
intermediates from the rest of the cell as in the
case of aflatoxin synthesis in Aspergillus parasiticus (Chanda et al. 2009). Little is known
about how SM biosynthetic pathways are compartmentalized in fungi, and it is unclear how
often this occurs. However, in cases where compartmentalization is important for product formation or to avoid toxicity effects, it is likely
essential that the pathway is organized in a
similar manner in a new host. Otherwise, it
may result in lack of production or even cell
death. In the future, we expect that pathway
compartmentalization will achieve significant
attention in SM cell factory construction. As a
standard control experiment, we advise to
determine whether individual enzymes in the
pathway localize to the same compartments in
the natural producer and in the new host, e.g.,
by GFP tagging. In cases where the host sorts an
enzyme incorrectly, it may be possible to
10 Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary. . .
251