Chemistry Reference
In-Depth Information
and alternaric acid (
52
), and it is thus an ideal target species for speculative PKS
gene-fishing expeditions. Fujii et al. (18) have conducted just such investigations,
using PCR primers based on conserved PKS sequences as probes with genomic
DNA libraries (18). An early investigation yielded two hits—one a HR PKS gene
named
alt5
, and another, a NR PKS named
pksA
.The
alt5
gene encodes a typical
HR PKS, which is known as PKSN, with the usual array of catalytic domains.
Inspection of the ER sequence suggested that it should be functional like those
from LDKS and SQTKS. Expression of
alt5
showed this to be correct—a single
compound was synthesized in good yield (
∼
15mgl
−
1
), which proved to be the
octamethylated decaketide pyrone (
53
), named alternapyrone. This compound is
the most complex polyketide yet reported to be produced by an iterative PKS,
which shows programmed chain length control, keto-reduction, methylation, and
enoyl reduction.
6.6.4 HR PKS-NRPS
Fungi produce a wide range of bioactive compounds derived from polyketides
fused to amino acids. Examples include fusarin C (
26
), equisetin (
54
), and tenellin
(
55
). Fusarin C (
26
) consists of a tetramethylated heptaketide fused to homoser-
ine and is produced by strains of the plant pathogens
Fusarium moniliforme
and
Fusarium venenatum
. Genomic DNA libraries from these organisms were used
to isolate a gene cluster centered around a 12-Kb ORF encoding a HR PKS fused
to a nonribosomal peptide synthetase (NRPS) module (19). The PKS region is
homologous to LNKS: KAS, AT, and DH domains are followed by CMeT, a
defective ER, KR, and ACP domains. Like LNKS, the ACP is upstream of an
NRPS condensation (C) domain, but in this case the NRPS module is complete,
which features downstream adenylation (A), thiolation (T), and C-terminal thi-
olester reductase (R) domains (Fig. 6.3c). Directed knockout of the PKS-NRPS
gene proved it to be involved in the biosynthesis of (
26
), and it was thus named
fusarin synthetase (FUSS). The disfunctional ER domain and the fact that no
lovC
homolog seems to exist in the cluster, is consistent with the polyunsaturated
nature of the polyketide moiety.
It is probable that FUSS assembles a tetramethylated heptaketide (
56
) attached
to the ACP (Fig. 6.3c)—the structure of which is similar to the heptaketide
pyrone (
46
) produced by LNKS in the absence of the LovC protein. In parallel, the
A domain of the NRPS module seems to select, activate, and attach homoserine
(
57
) to the thiolation domain. The C domain then uses the amide of homoserine to
form an amide with the ACP-bound polyketide, which forms a covalently bound
intermediate peptide (
58
). The final reaction catalyzed by FUSS may be reductive
release of the thiolester, which forms peptide aldehyde (
59
). Finally, Knoevenagel
cyclization would give the putative prefusarin (
60
). Other genes in the FUSS
cluster are presumably responsible for the required additional transformation of
(
60
)tofusarinC(
26
) itself: epoxidation; oxidation of a pendant methyl to a
carboxylate and esterification; and hydroxylation a to nitrogen.
