Download Sorting the Fatty Acid Chaff from the Toxin Wheat, or is it All

Proceedings 15th ICHA, accepted manuscript
Sorting the Fatty Acid Chaff from the Toxin Wheat, or is it All Wheat? Assigning Dinoflagellate PKS genes to Toxin Synthesis
Allen Place
Institute of Marine and Environmental Technology, University of Maryland Center for Environmental
Sciences, 701 E. Pratt Street, Baltimore, MD, USA 21202, [email protected]
Abstract
Success in identifying genes and enzymes that are involved in the biosynthesis of toxins by dinoflagellates
has been limited thus far, despite considerable efforts by many groups. The chemical structures of
dinoflagellate polyketides suggest that they are produced by modular type I PKS enzymes in some cases with
an involvement of a NRPS, for instance in the case of DTX-5a/5b and spirolides. Unfortunately,
dinoflagellates also make fatty acids using PKS machinery so it is difficult to discern the machinery involved
in toxin synthesis from those involved in fatty acid synthesis. However, we believe there are several research
avenues that can be pursued to open the door to this unique biosynthetic machinery. Given the light
dependency of karlotoxin production we hypothesize the starter unit for karlotoxin and amphidinol
biosynthesis is glycolate that comes from photorespiration. We argue that the acyl carrier protein and acyl
transferase (loader) will be different from that used in fatty acid synthesis because the starter unit is different
(i.e. glycolate vs acetate). Focusing only on the acyl carrier proteins and acyl transferases we have found two
candidate proteins that we believe are involved in the initiation of karlotoxin synthesis.
Keywords: karlotoxin, polyketides, dinoflagellate, biosynthesis
Introduction
The chemical structures of polyketides from
dinoflagellates suggest that they are produced by
type I polyketide synthases (PKSs), and in some
cases with the involvement of a non-ribosomal
peptide synthetase (NRPSs). A minimal PKS has
an acyltransferase (AT) domain, a β-ketosynthase(KS) domain, and an acyl carrier protein (ACP).
The AT domain covalently transfers a specific
carboxylic acid from acyl-CoA to the ACP, which
is then condensed by the KS domain to another
ACP-bound acyl chain. A PKS module may have
optional β-ketoacyl reductase (KR), dehydrogenase
(DH), and enoyl reductase (ER) domains, which
reduce the β-ketone to an alcohol, dehydrate the
alcohol, and saturate the resultant double bond,
respectively. In analogy, a minimal NRPS provides
an adenylation domain (A), which specifically
activates an amino acid, a peptidyl carrier protein
(PCP), and a condensation domain (C) that creates
a peptide bond between two PCP-bound amino
acids. Thioesterase (TE) domains may release, and
cyclize the final enzyme products.
Type I PKSs and NRPSs usually consist of large,
non-iterative, multidomain enzymes. Modular type
I PKSs and NRPSs form megasynthetases that
generally follow a colinearity rule, where one
module extends a growing acyl or peptidyl chain
by one particular unit. Each PKS module carries a
set of catalytic domains that perform one round of
polyketide elongation and modification. During
these processes, an AT domain attaches the
corresponding acyl-CoA building block onto an
acyl carrier protein domain (ACP), a KS domain
elongates the polyketide chain with this acyl unit
and optional additional domains further modify the
resulting intermediate. An example of this modular
synthesis is presented in Fig. 1 for the first 14
carbons of karlotoxin with glycolate being the
starter unit rather than acetate. For these 14
carbons, 8 KS and AT domains are required with
only 7 KRs and 3 and 2 DHs and ERs modules.
However, these 8 modular genes have not been
found in any karlotoxin producing species.
The frequently observed close correlation between
the domain architecture and the sequence of
functional groups in the polyketide chain, codified
as the ‘colinearity rule’, has enabled direct
prediction of polyketide structures from genomic
sequences and vice versa. However, this
colinearity appears to be broken in dinoflagellates.
Kubota et al. (2006) screened genomic DNA from
five amphidinolide-producing and eight nonproducing dinoflagellate strains by degenerate
PCR for the presence of β-ketosynthase (KS)
domains of type I PKS genes. Fragments of
Proceedings 15th ICHA, accepted manuscript
Fig. 1. Predicted PKS modular structure for the
first 14 carbons of karlotoxin. The starter unit was
glycolate rather than acetate.
fourteen unique KS domains were detected. These
sequences
were
exclusively
present
in
amphidinolide producer strains, and a genomic
fosmid DNA library was constructed from the
amphidinolide-producing strain Amphidinium sp.
Y-42. Kubota et al. (2006) detected a single clone
out of a total of 100,000 PCR-screened clones,
which harbored PKS-related sequences, and the
entire fosmid insert (36.4 kb) was sequenced. The
fosmid insert had six sequence regions, KS, AT,
DH, KR, ACP, and TE that were related to type I
PKS genes. Their genomic arrangement was
unusual however, as several frame-shifts occurred
within and between catalytic domains. The proteincoding region was flanked on both sides by long
stretches of non-coding sequence, and the mid
section of the protein-coding region contained a 4
kb stretch of sequence that presumably represented
an intron. Only approximately 15% of the 36.4 kb
long fosmid insert consisted of protein-coding
sequence. This sequence encoded putative catalytic
functions for only a single elongation cycle of a
26-membered polyketide. If one would extrapolate
based on these data, all genes required for the
production of amphidinolide may occupy up to 500
kb of genomic DNA. Further, it would not be
certain, whether they were present on the same
locus, or distributed throughout the genome. This
study exemplifies the huge challenges associated
with characterizing biosynthesis genes in
dinoflagellates on the genomic level, regardless of
the sequencing technology used. Unfortunately,
sequences obtained in the study by Kubota et al.
(2006) were not deposited in GenBank, preventing
further analysis. Similarly, when Bachvaroff and
Place (2008) tried to find other modules of a KR
gene in A. carterae no other PKS modules were
found within 12 kb of genomic DNA. This gene
was also interspersed with numerous introns. And
lastly, when Monroe and Van Dolah (2008)
characterized the PKS cDNAs from K. brevis they
only found transcripts that contained one catalytic
module rather than multiple modules expected for
a modular Type I PKS transcript.
Recently a novel group of modular PKSs that
diverge from the canonical (cis-AT) type
architecture have been described (Nguyen 2008).
These trans-AT PKSs are characterized by the
absence of an integrated AT domain in each
module which is complemented by free-standing
acyl transferases. Phylogenetic data suggest that
these trans-AT PKSs evolved independently from
cis-AT PKSs. We believe this is the mode of
polyketide synthesis in dinoflagellates. In addition
to the unique acyl transfer mechanism, the
enzymes are noteworthy for exhibiting highly
aberrant architectures with modules carrying novel
catalytic domains or domain orders, or having no
apparent function or relation to polyketide
structure. These could only be compared using
bioinformatic approaches of the amino acid
sequences.
Approach
Given the light dependency of karlotoxin
production we hypothesize the starter unit for
karlotoxin and amphidinol biosynthesis is
glycolate that comes from photorespiration. A
previous study by Murata’s group established the
polyketide origin of three amphidinols, AM2, AM3
and AM4 (Houdai et al. 2001) using 13C labeled
acetate. The enrichment patterns observed revealed
that the carbon chain of AM17 is derived entirely
from acetate units, including the pendant carbon
atoms. Both C-1 and C-2 of the chain were
however, unlabeled, similar to a result obtained for
other amphidinols, and this portion of the chain
was inferred to result from a glycolate starter unit
as observed in okadaic acid and its analogs (Wright
et al. 1996). Recently, glycolate was shown to be
the starter unit for yessotoxin synthesis (Yamazaki
et al., 2010) and adding glycolate to the culture
media
enhances
gymnodimine
production
(Mountfort et al., 2006). This is currently being
tested with feeding studies on K. veneficum and A.
carterae.
Proceedings 15th ICHA, accepted manuscript
Fig. 2. Predicted domains for a plastid acyl carrier protein cDNA from K. veneficum (CCMP 2778)
Fig. 3. Predicted domains for a CA-Acyl Carrier Protein Transacylase cDNA from K. veneficum (CCMP
2778).
From the current annotation of the full-length
cDNA libraries (>160,000) of K. veneficum and A.
carterae (NSF Microbial Genome Sequencing
Program grant #EF-0626678, “Dinoflagellate fulllength cDNA sequencing”) we have already
identified greater than 200 genes potentially
involved in fatty acid and karlotoxin (or
amphidinol) synthesis. To sort through these
candidate genes we have focused on the initiation
of the PKS cycle looking for enzymes that might
transfer glycolate to an acyl carrier protein and
could also accommodate a large growing
polyketide chain. We have found two candidate
proteins that we believe are involved in the
initiation of karlotoxin synthesis. The first (Fig. 2)
is a large acyl carrier protein (ACP) (359 amino
acids) that differs significantly from the smaller
ACPs (~134 amino acids). This protein has a
chloroplast targeting sequence. We envision the
numerous smaller cytoplasmic ACPs are involved
in fatty acid synthesis while the larger plastid
protein receives the starter glycolate for karlotoxin
synthesis. The second candidate is a malonyl
carrier protein transacylase (Fig. 3) that is
Proceedings 15th ICHA, accepted manuscript
phylogenetically distinct from the other CoA-acyl
carrier protein transacylases found in the libraries.
We envision this transacylase charges the ACP
with glycolate in the chloroplast. We are currently
testing these hypotheses using antibodies to each
of these proteins.
Conclusion
Only by focusing on the initiation of the PKS cycle
can we sort the metabolic players involved in toxin
biosynthesis from those involved in fatty acid
synthesis. Even if this approach proves successful
it is highly likely that many of the same players
may be involved in both processes.
Acknowledgements:
This work was supported by the NOAA PCM grant
# NA10NOS4780154. This is contribution #4 from
NOAA PCM program, #4875 from the University
of Maryland Center for Environmental Sciences,
and contribution #145 from the Institute of Marine
and Environmental Technology.
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