ensure proper localization of the relevant
enzyme by fusing it to transport signals or a
carrier protein that is sorted to the correct
compartment.
B. Secondary Metabolite Discovery via
Different Gene-Expression Systems
Multiple gene-expression strategies and different fungal hosts have been used for SMs discovery and pathway elucidation by heterologous
gene expression. A. oryzae, unlike most other
common fungal cell factories, produces few
SMs. A. oryzae is therefore often chosen as a
host as the subsequent detection of the new SM
in the metabolite profile is relatively simple.
However, with A. oryzae genetic engineering is
complicated by the fact that its asexual spores
contain several nuclei (Maruyama et al. 2001),
which makes purification of transformed
strains more laborious. In contrast, A. nidulans
produces asexual spores that contain only a
single nucleus (Yuill 1950), and transformants
are therefore relatively easy to purify. Hence, if
pathway elucidation requires more elaborate
genetic engineering, species like A. nidulans
may be a better choice of host. Moreover, in
some cases, the more complex host-chemistry
can advantageously contribute to formation of
novel synthetic and potentially useful compounds. Alternatively, it may facilitate construction of a new synthetic pathway by
delivering a missing activity in the desired
pathway. Below we will provide successful
examples of heterologous SM production
using different hosts and expression systems,
which may serve as an inspiration for construction of new fungal SM cell factories.
1. Heterologous Expression of Secondary
Metabolite Synthase Genes
To investigate whether a new SM can be produced in a given cell factory, it may often be
advantageous to establish the first step of a
multi-enzyme pathway, which produces the
scaffold of the final compound. Indeed, the
first demonstration of a heterologously produced SM was 6-methylsalicylic acid (6-MSA)
(Fujii et al. 1996), which serves as a precursor of
several SMs including patulin and yanuthones
(Beck et al. 1990; Holm et al. 2014; Petersen
et al. 2015; Read and Vining 1968). Hence, it is
of interest to develop efficient systems for single gene transfer. In A. oryzae, a plasmid was
constructed to allow a gene to be inserted
ectopically into the genome in multiple copies
(Fujii et al. 1995).
This plasmid has been widely applied for heterologous
gene expression, and it has been used to deliver basic
SM scaffolds including production of the naphthopyrone YWA1 (Watanabe et al. 1998, 1999), alternapyrone
(Fujii et al. 2005), ferrirhodin (Munawar et al. 2013),
and astellifadiene (Matsuda et al. 2016).
In A. nidulans, where genetic engineering is
easier, genes have often been inserted into a
defined locus to facilitate strain characterization and to obtain better gene-expression control. To ensure high expression levels, it may be
useful to position integration sites in intergenic
regions located in transcriptionally highly
active sections of a chromosome.
Using this approach, it has been demonstrated that
MpaC from P. brevicompactum produces 5methylorsellinic acid (5-MOA) as the first intermediate
toward production of the immunosuppressant drug
mycophenolic acid by expressing mpaC in A. nidulans
(Hansen et al. 2011b).
The same setup was used to make a cell
factory yielding 1.8 g/l of 6-MSA by expressing
yanA from A. niger in A. nidulans (Knudsen
2015), which is sixfold higher as compared to
ectopic integration in A. oryzae reported by
Fujii et al. (1996). Using the A. nidulans 6MSA cell factory, it was possible to produce
13
C-labelled 6-MSA, which was subsequently
used to clarify the biosynthetic pathway for
yanuthone D production in A. niger (Holm
et al. 2014). The wA locus of A. nidulans has
also been used as a gene-expression site. In this
case, correct transformants can easily be identified as they produce white conidia. Using this
approach, Chiang and co-workers discovered
several new compounds by expressing A. terreus polyketide synthase genes in A. nidulans
(Chiang et al. 2013).
252
J. K. H. Rendsvig et al.
enzyme by fusing it to transport signals or a
carrier protein that is sorted to the correct
compartment.
B. Secondary Metabolite Discovery via
Different Gene-Expression Systems
Multiple gene-expression strategies and different fungal hosts have been used for SMs discovery and pathway elucidation by heterologous
gene expression. A. oryzae, unlike most other
common fungal cell factories, produces few
SMs. A. oryzae is therefore often chosen as a
host as the subsequent detection of the new SM
in the metabolite profile is relatively simple.
However, with A. oryzae genetic engineering is
complicated by the fact that its asexual spores
contain several nuclei (Maruyama et al. 2001),
which makes purification of transformed
strains more laborious. In contrast, A. nidulans
produces asexual spores that contain only a
single nucleus (Yuill 1950), and transformants
are therefore relatively easy to purify. Hence, if
pathway elucidation requires more elaborate
genetic engineering, species like A. nidulans
may be a better choice of host. Moreover, in
some cases, the more complex host-chemistry
can advantageously contribute to formation of
novel synthetic and potentially useful compounds. Alternatively, it may facilitate construction of a new synthetic pathway by
delivering a missing activity in the desired
pathway. Below we will provide successful
examples of heterologous SM production
using different hosts and expression systems,
which may serve as an inspiration for construction of new fungal SM cell factories.
1. Heterologous Expression of Secondary
Metabolite Synthase Genes
To investigate whether a new SM can be produced in a given cell factory, it may often be
advantageous to establish the first step of a
multi-enzyme pathway, which produces the
scaffold of the final compound. Indeed, the
first demonstration of a heterologously produced SM was 6-methylsalicylic acid (6-MSA)
(Fujii et al. 1996), which serves as a precursor of
several SMs including patulin and yanuthones
(Beck et al. 1990; Holm et al. 2014; Petersen
et al. 2015; Read and Vining 1968). Hence, it is
of interest to develop efficient systems for single gene transfer. In A. oryzae, a plasmid was
constructed to allow a gene to be inserted
ectopically into the genome in multiple copies
(Fujii et al. 1995).
This plasmid has been widely applied for heterologous
gene expression, and it has been used to deliver basic
SM scaffolds including production of the naphthopyrone YWA1 (Watanabe et al. 1998, 1999), alternapyrone
(Fujii et al. 2005), ferrirhodin (Munawar et al. 2013),
and astellifadiene (Matsuda et al. 2016).
In A. nidulans, where genetic engineering is
easier, genes have often been inserted into a
defined locus to facilitate strain characterization and to obtain better gene-expression control. To ensure high expression levels, it may be
useful to position integration sites in intergenic
regions located in transcriptionally highly
active sections of a chromosome.
Using this approach, it has been demonstrated that
MpaC from P. brevicompactum produces 5methylorsellinic acid (5-MOA) as the first intermediate
toward production of the immunosuppressant drug
mycophenolic acid by expressing mpaC in A. nidulans
(Hansen et al. 2011b).
The same setup was used to make a cell
factory yielding 1.8 g/l of 6-MSA by expressing
yanA from A. niger in A. nidulans (Knudsen
2015), which is sixfold higher as compared to
ectopic integration in A. oryzae reported by
Fujii et al. (1996). Using the A. nidulans 6MSA cell factory, it was possible to produce
13
C-labelled 6-MSA, which was subsequently
used to clarify the biosynthetic pathway for
yanuthone D production in A. niger (Holm
et al. 2014). The wA locus of A. nidulans has
also been used as a gene-expression site. In this
case, correct transformants can easily be identified as they produce white conidia. Using this
approach, Chiang and co-workers discovered
several new compounds by expressing A. terreus polyketide synthase genes in A. nidulans
(Chiang et al. 2013).
252
J. K. H. Rendsvig et al.