88
H. Zhu et al.
4.2.1 The Early Stages of Cytochalasan Biosynthesis
To validate the early formation mechanism of cytochalasan, an exploratory study on
the biosynthesis of cytochalasin E (145) was conducted by Oikawa and co-workers.
They co-expressed heterologously the genes encoding the PKS–NRPS, ccsA, and
the trans-ER, ccsC from A. clavatus together in Aspergillus oryzae [262]. Unexpectedly, neither the supposed aldehyde nor pyrrolinone structure was obtained when
these genes were overexpressed, instead, an octaketide alcohol A1 intermediate was
isolated and identified (Scheme 2a). Another unexpected feature was the absence
of an olefin between C-4 and C-5 on the polyketide chain, which is observed in
cytochalasin E (145), suggesting a reduction reaction catalyzed by an enzyme within
A. oryzae [2] might take place on the backbone.
A similar type of investigation has been conducted by Larsen and coworkers,
who used Aspergillus nidulans as the heterologous expression host to characterize
the functions of ccsA and ccsC [252]. They found that the expression of ccsA alone
did not yield any detectable products, and the expression of trans-ER ccsC along
with ccsA was vital for the product formation. The A. nidulans strain co-expressed
ccsA and ccsC and produced several products, of which a minor one was speculated
to be identical to the previously discovered intermediate A1 by Oikawa’s group,
as the same compound mass at 440.3188 Da was measured. Differing from A1, a
linear PKS-NRPS precursor, the major product named niduclavin (A2) (Scheme 2A),
possesses a phenylalanine moiety joining a decalin scaffold that originates from a
highly reduced octaketide chain. In comparison to the general structure of cytochalasans such as cytochalasin E (145), niduclavin (A2) has an additional double bond
between the C-2
and C-3
positions of the phenylalanine side chain. The authors speculated that cross-chemical reactions with endogenous enzymes from A. nidulans were
responsible for the introduction of the double bond, which would additionally activate
the dienophile in the α/β/-position (C-4 and C-5) of the C-3 carbonyl group, thereby
possibly favoring decalin formation rather than the tetramic acid moiety present in
the native A. clavatus molecule. Inactivation of a dioxygenase AsqJ, suspected to be
responsible for the introduction of the C-2
/C-3
double bond, did not prevent the
formation of niduclavin (A2). The genes CcsA and ccsC had also been expressed in
an alternate heterologous host, Aspergillus niger. Co-expression of these two genes
in A. niger also led to the production of niduclavin (A2) [252].
As the previous basic local alignment search tool research (BLAST) revealed a
high degree of similarity between CcsA and the SYN2 in the ACE1 cluster (68%
similarity and 52% identity). Larsen’s group studied the cryptic genes SYN2 and
RAP2 present in the ACE1 gene cluster (Fig. 21A) from M. oryzae in the same
study [252]. Analogous to the integration and expression of ccsA and ccsC, SYN2
and RAP2 were transformed into A. nidulans. The SYN2/RAP2 product was purified
and a structure with a close resemblance to niduclavin A2 was determined by NMR
spectroscopy and given the name niduporthin A3 (Scheme 2B). Key differences
between niduporthin A3 and niduclavin A2 include incorporation of different amino
acid and methyl group substitutions on the polyketide chain.
H. Zhu et al.
4.2.1 The Early Stages of Cytochalasan Biosynthesis
To validate the early formation mechanism of cytochalasan, an exploratory study on
the biosynthesis of cytochalasin E (145) was conducted by Oikawa and co-workers.
They co-expressed heterologously the genes encoding the PKS–NRPS, ccsA, and
the trans-ER, ccsC from A. clavatus together in Aspergillus oryzae [262]. Unexpectedly, neither the supposed aldehyde nor pyrrolinone structure was obtained when
these genes were overexpressed, instead, an octaketide alcohol A1 intermediate was
isolated and identified (Scheme 2a). Another unexpected feature was the absence
of an olefin between C-4 and C-5 on the polyketide chain, which is observed in
cytochalasin E (145), suggesting a reduction reaction catalyzed by an enzyme within
A. oryzae [2] might take place on the backbone.
A similar type of investigation has been conducted by Larsen and coworkers,
who used Aspergillus nidulans as the heterologous expression host to characterize
the functions of ccsA and ccsC [252]. They found that the expression of ccsA alone
did not yield any detectable products, and the expression of trans-ER ccsC along
with ccsA was vital for the product formation. The A. nidulans strain co-expressed
ccsA and ccsC and produced several products, of which a minor one was speculated
to be identical to the previously discovered intermediate A1 by Oikawa’s group,
as the same compound mass at 440.3188 Da was measured. Differing from A1, a
linear PKS-NRPS precursor, the major product named niduclavin (A2) (Scheme 2A),
possesses a phenylalanine moiety joining a decalin scaffold that originates from a
highly reduced octaketide chain. In comparison to the general structure of cytochalasans such as cytochalasin E (145), niduclavin (A2) has an additional double bond
between the C-2
and C-3
positions of the phenylalanine side chain. The authors speculated that cross-chemical reactions with endogenous enzymes from A. nidulans were
responsible for the introduction of the double bond, which would additionally activate
the dienophile in the α/β/-position (C-4 and C-5) of the C-3 carbonyl group, thereby
possibly favoring decalin formation rather than the tetramic acid moiety present in
the native A. clavatus molecule. Inactivation of a dioxygenase AsqJ, suspected to be
responsible for the introduction of the C-2
/C-3
double bond, did not prevent the
formation of niduclavin (A2). The genes CcsA and ccsC had also been expressed in
an alternate heterologous host, Aspergillus niger. Co-expression of these two genes
in A. niger also led to the production of niduclavin (A2) [252].
As the previous basic local alignment search tool research (BLAST) revealed a
high degree of similarity between CcsA and the SYN2 in the ACE1 cluster (68%
similarity and 52% identity). Larsen’s group studied the cryptic genes SYN2 and
RAP2 present in the ACE1 gene cluster (Fig. 21A) from M. oryzae in the same
study [252]. Analogous to the integration and expression of ccsA and ccsC, SYN2
and RAP2 were transformed into A. nidulans. The SYN2/RAP2 product was purified
and a structure with a close resemblance to niduclavin A2 was determined by NMR
spectroscopy and given the name niduporthin A3 (Scheme 2B). Key differences
between niduporthin A3 and niduclavin A2 include incorporation of different amino
acid and methyl group substitutions on the polyketide chain.