Progress in the Chemistry of Cytochalasans
83
4.1 Cytochalasan Gene Clusters
The first cytochalasan gene cluster reported in 2004 was ACE1 identified from
Magnaporthe oryzae Guy11 (named earlier Magnaporthe grisea), and since then has
been further investigated [248–250]. The ACE1 gene cluster was speculated to confer
a virulence factor that is recognized by rice cultivars carrying the resistance gene Pi33,
enabling the rice to activate a defensive response. This cluster encodes several biosynthesis proteins, which were annotated as two PKS–NRPS (ACE1 and SYN2), two
trans-ER proteins (RAP1 and RAP2), four P450 monooxygenases (CYP1-4), two
FAD-dependent monooxygenases (OXR1 and OXR2), an α,β-hydrolase (ORFZ), a
putative Diels–Alderase (pDA, ORF3), a transporter (MFS) and a regulator (BC2)
(Fig. 21A).
Investigation into the evolution of the ACE1 gene cluster by Wolfe and co-workers
indicated that it is distributed sporadically and shared by only a few fungal species
[250]. The evolution of the ACE1-like gene clusters is characterized by complex
events including gene duplication and losses, gene recruitment, and horizontal gene
transfer [250]. Phylogenetic analysis has indicated that the ACE1 cluster is related to
the gene clusters in Chaetomium globosum and Aspergillus clavatus, which were later
shown to encode cytochalasan molecules. It was also suggested that in Magnaporthe
oryza, the physical linkage between the three core genes, PKS-NRPS (ACE1), transER (RAP1) and pDA (ORF3) e, is ancient, and can be inferred to have existed in a
common ancestor [250].
Although the secondary metabolite(s) from the ACE1 gene cluster has not yet
been isolated, due to their tightly regulated temporal expression, recent work by two
independent groups suggests that the ACE1 gene cluster encodes the biosynthesis of
two different cytochalasan compounds [251, 252].
The first cytochalasan gene cluster to be characterized was the chaetoglobosin
A (208) (che) cluster (Fig. 21B) from Penicillium expansum [253]. Before this
work, numerous isotope labeling experiments suggested that cytochalasan biosynthesis involves the formation of acetate and methionine-derived octa- or nonaketide
chain and the attachment of an amino acid, which was referred to as the PKS-NRPS
pathway in the biosynthesis of other secondary metabolites such as fusarin [254],
equisetin [255], tenellin [256], and aspyridone [257]. In order to discern the molecular basis of cytochalasan biosynthesis, Hertweck and coworkers used a heterologous probe spanning the C-methyltransferase (CMeT), ketoreductase (KR), and acyl
carrier protein (ACP) domain coding regions from the Fusarium venenatum fusarin
C PKS–NRPS, to screen a Penicillium expansum cosmid library. As a result, the che
gene cluster consisted of seven genes encoding a PKS–NRPS (CheA), a trans enoylreductase (CheB) [258], two putative P450 monooxygenases (CheD and CheG), a
FAD-dependent monooxygenase (CheE), and two transcription factors (CheC and
CheF). Bioinformatic analysis indicated that the gene cluster was most similar to the
aspyridone cluster in A. nidulans. As all attempts to generate a targeted gene knockout in P. expansum and heterologous expression of the entire or partial gene cluster in
Aspergillus failed, the cluster was confirmed putatively, using RNA-mediated gene
83
4.1 Cytochalasan Gene Clusters
The first cytochalasan gene cluster reported in 2004 was ACE1 identified from
Magnaporthe oryzae Guy11 (named earlier Magnaporthe grisea), and since then has
been further investigated [248–250]. The ACE1 gene cluster was speculated to confer
a virulence factor that is recognized by rice cultivars carrying the resistance gene Pi33,
enabling the rice to activate a defensive response. This cluster encodes several biosynthesis proteins, which were annotated as two PKS–NRPS (ACE1 and SYN2), two
trans-ER proteins (RAP1 and RAP2), four P450 monooxygenases (CYP1-4), two
FAD-dependent monooxygenases (OXR1 and OXR2), an α,β-hydrolase (ORFZ), a
putative Diels–Alderase (pDA, ORF3), a transporter (MFS) and a regulator (BC2)
(Fig. 21A).
Investigation into the evolution of the ACE1 gene cluster by Wolfe and co-workers
indicated that it is distributed sporadically and shared by only a few fungal species
[250]. The evolution of the ACE1-like gene clusters is characterized by complex
events including gene duplication and losses, gene recruitment, and horizontal gene
transfer [250]. Phylogenetic analysis has indicated that the ACE1 cluster is related to
the gene clusters in Chaetomium globosum and Aspergillus clavatus, which were later
shown to encode cytochalasan molecules. It was also suggested that in Magnaporthe
oryza, the physical linkage between the three core genes, PKS-NRPS (ACE1), transER (RAP1) and pDA (ORF3) e, is ancient, and can be inferred to have existed in a
common ancestor [250].
Although the secondary metabolite(s) from the ACE1 gene cluster has not yet
been isolated, due to their tightly regulated temporal expression, recent work by two
independent groups suggests that the ACE1 gene cluster encodes the biosynthesis of
two different cytochalasan compounds [251, 252].
The first cytochalasan gene cluster to be characterized was the chaetoglobosin
A (208) (che) cluster (Fig. 21B) from Penicillium expansum [253]. Before this
work, numerous isotope labeling experiments suggested that cytochalasan biosynthesis involves the formation of acetate and methionine-derived octa- or nonaketide
chain and the attachment of an amino acid, which was referred to as the PKS-NRPS
pathway in the biosynthesis of other secondary metabolites such as fusarin [254],
equisetin [255], tenellin [256], and aspyridone [257]. In order to discern the molecular basis of cytochalasan biosynthesis, Hertweck and coworkers used a heterologous probe spanning the C-methyltransferase (CMeT), ketoreductase (KR), and acyl
carrier protein (ACP) domain coding regions from the Fusarium venenatum fusarin
C PKS–NRPS, to screen a Penicillium expansum cosmid library. As a result, the che
gene cluster consisted of seven genes encoding a PKS–NRPS (CheA), a trans enoylreductase (CheB) [258], two putative P450 monooxygenases (CheD and CheG), a
FAD-dependent monooxygenase (CheE), and two transcription factors (CheC and
CheF). Bioinformatic analysis indicated that the gene cluster was most similar to the
aspyridone cluster in A. nidulans. As all attempts to generate a targeted gene knockout in P. expansum and heterologous expression of the entire or partial gene cluster in
Aspergillus failed, the cluster was confirmed putatively, using RNA-mediated gene