Scalable Biosynthesis of the Seaweed Neurochemical, Kainic Acid

Article abstract of DOI:10.1002/anie.201902910

Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuropharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six-steps. Herein we report the discovery and characterization of the concise two-enzyme biosynthetic pathway to kainic acid from l-glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co-clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α-ketoglutarate-dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.

Full text of DOI:10.1002/anie.201902910

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Scalable biosynthesis of the seaweed neurochemical kainic acid
Authors: Jonathan R. Chekan, Shaun M. K. McKinnie, Malia L. Moore,
Shane G. Poplawski, Todd P. Michael, and Bradley S. Moore
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201902910
Angew. Chem. 10.1002/ange.201902910
Link to VoR: http://dx.doi.org/10.1002/anie.201902910
http://dx.doi.org/10.1002/ange.201902910

10.1002/anie.201902910
Angewandte Chemie International Edition
COMMUNICATION
Scalable biosynthesis of the seaweed neurochemical kainic acid
Jonathan R. Chekan[a], Shaun M. K. McKinnie[a], Malia L. Moore[a], Shane G. Poplawski[b], Todd P.
Michael[b], & Bradley S. Moore[a],[c]*
Abstract: Kainic acid, the flagship member of the kainoid family of
natural neurochemicals, is a widely used neuropharmacological agent
that helped unravel the key role of ionotropic glutamate receptors,
including the kainate receptor, in the central nervous system.
Worldwide shortages of this seaweed natural product in 2000
prompted numerous chemical syntheses that now number in excess
of 70, including scalable preparations with as few as six-steps. Herein
we report the discovery and characterization of the concise two-
enzyme biosynthetic pathway to kainic acid from L-glutamic acid and
dimethylallyl pyrophosphate in red macroalgae and show that the
Figure 1. Notable synthetic routes to kainic acid by (A) Fukuyama,15 (B)
biosynthetic genes are co-clustered in genomes of Digenea simplex
Ohshima,16 and (C) Baldwin17 and coworkers.
and the edible Palmaria palmata. Moreover, we applied a key
ene-cyclization,[16] or radical formation.[17] Unfortunately,
biosynthetic α-ketoglutarate-dependent dioxygenase enzyme in a
challenges in generating the three contiguous stereocenters often
limit these approaches to low yields or many steps. In contrast to
the synthetic work, little progress has been made on elucidating
how kainic acid is constructed by seaweeds. Recently, we
established the biosynthetic logic for domoic acid production in
microalgal Pseudo-nitzschia multiseries diatoms through the
discovery of a four-gene cassette (dabA-D) and the confirmation
of their in vitro enzymatic functions (Figure 2A).[18] The structural
similarity between domoic acid and kainic acid allowed us to
propose a conserved route of biosynthesis (Figure 2B). N-
prenylation of L-glutamate with dimethylallyl pyrophosphate
(DMAPP) via a DabA homolog would produce a “prekainic acid”
pathway intermediate that could conceivably be cyclized directly
with a DabC homolog to generate kainic acid.
biotransformation methodology to efficiently construct kainic acid on
the gram scale. This study establishes both the feasibility of mining
seaweed genomes for their biotechnological prowess and the
applicability of the genes as biocatalysts in the synthesis of fine
chemicals.
The tropical seaweed Digenea simplex has been used for
centuries in Asia as an anthelmintic agent to treat parasitic worm
infections.[1] In the 1950s, the active compound, kainic acid, was
isolated,[2,3] enabling its use as a combination treatment for
Ascaris infections up until the 1990s.[4–6] While the exact
anthelmintic mechanism of action remains unclear, kainic acid
functions as an ionotropic glutamate receptor (iGluR) agonist.[7]
iGluRs mediate neuronal cell-cell communication by binding to
glutamate and facilitating influx of Ca2+ into the cell.[8] Structurally
similar to glutamic acid, kainic acid can bind more efficiently to
iGluRs and thereby stimulate excessive influx of Ca2+, which
leads to excitotoxicity and cell death.[9] In fact, kainic acid was
important in the initial discovery and characterization of several
classes of iGluRs, such as the eponymous kainate receptor,[10]
and has been exploited to create mouse model systems to study
neurological diseases,[11] particularly temporal lobe epilepsy.[12,13]
In the 50 years since kainic acid was isolated, over 70 synthetic
routes have been established (Figure 1).[14] These syntheses
employ diverse methods to produce the kainoid ring
pharmacophore such as ring opening followed by cyclization,[15]
To validate our hypothesis, we performed whole genome
sequencing of the well-studied kainic acid producer D. simplex
from Japan to identify the kainic acid biosynthetic genes and
determine their genetic context. Red macroalgal genomes have
proven challenging due to their close association with a diverse
array of marine microbes and crustaceans, which means
sequencing efforts result in highly complex metagenomes if they
are not cured with antibiotics.[19] Moreover, identifying biosynthetic
cassettes in red macroalgae genomes, which are often tandem
duplications and nested in repetitive sequence, is almost
impossible with current high throughput short read (~ 100 bp)
sequencing technologies. However, the new single molecule long
read sequencing platforms, such as Oxford Nanopore
Technologies (ONT), generate single reads in the hundreds of
kilobases (kb), which greatly facilitates resolving tandemly
duplicated genes and repeats, assembling metagenomes, and
generating highly contiguous reference genomes.[20] We
sequenced D. simplex on the ONT MinION platform and
produced 7 Gb of sequence with a read N50 of 7 kb and the
longest read at 235 kb. We were able to find long reads that
contained genes suggestive of kainic acid biosynthesis, but due
to the amount of microbial sequence we were not able to
assemble the D. simplex genome. Therefore, we sequenced D.
simplex on the higher throughput ONT PromethION platform and
generated 47 Gb of sequence with a read N50 of 7 kb and the
longest read at 1.2 Mb (Figure S1). The resulting correction-less
[a]
Dr. J. R. Chekan, Dr. S. M. K. McKinnie, M. L. Moore, Prof. Dr. B. S.
Moore
Center for Marine Biotechnology and Biomedicine
Scripps Institution of Oceanography
University of California, San Diego, La Jolla, CA 92093 (USA)
E-mail: bsmoore@ucsd.edu
[b]
[c]
Dr. S. G. Poplawski, Dr. T. P. Michael
J. Craig Venter Institute, La Jolla, CA 92037, USA.
Prof. Dr. B. S. Moore
Skaggs School of Pharmacy and Pharmaceutical Sciences
University of California, San Diego, La Jolla, CA 92093 (USA)
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201902910
Angewandte Chemie International Edition
COMMUNICATION
KabC αKG-dependent dioxygenase proteins revealed high levels
of conservation (Figures S3 and S4).
To confirm the proposed kainic acid biosynthetic pathway,
dskabA and dskabC were successfully expressed in Escherichia
coli and purified to homogeneity (Figure S5). Incubation of
DsKabA with the anticipated substrates DMAPP and L-glutamate
produced N-dimethylallyl-L-glutamic acid (prekainic acid) as
confirmed by NMR structural elucidation and LCMS comparison
with a synthetic standard (Figure 3). Consistent with other
members of the terpene cyclase family fold, Mg2+ is a required
cofactor in the reaction. We next explored the substrate selectivity
of DsKabA. Exchange of L-glutamate for D-glutamate showed
diminished turnover while L-glutamine, L-aspartate, L-asparagine,
and glycine did not produce appreciable amounts of N-prenylated
products (Figure S6). In contrast, when we replaced DMAPP with
GPP in the L-glutamate reaction, we clearly observed the
formation of N-geranyl-L-glutamic acid (L-NGG), the first
biosynthetic precursor to domoic acid (Figure S6). This high level
of amino acid selectivity and modest prenyl donor promiscuity was
also observed with DabA from domoic acid biosynthesis.[18] To
support the physiological relevance of prekainic acid in the
biosynthesis of kainic acid, we examined the aqueous extract of
D. simplex. High resolution LCMS analysis revealed the presence
of a compound with both the exact mass and retention time of
prekainic acid (Figure S7).
Figure 2. The proposed (A) domoic acid18 and (B) kainic acid biosynthetic
pathways. The kainic acid biosynthesis (kab) gene cluster from (C) D. simplex
and (D) P. palmata. Scale bar is representative for both (C) and (D).
genome assembly was 299 Mb with 8,246 contigs. The contig
N50 was 57 kb and the longest contig was 3.9 Mb. Based on GC%
analysis, we predict 205 Mb of the assembly is D. simplex, while
88 Mb is high GC% bacterial contigs. This assembly was
subsequently polished with Illumina short-read sequencing.
Upon analyzing the D. simplex genome for the presence of
homologs to the domoic acid biosynthetic (dab) genes, we
uncovered a 12 kb kainic acid biosynthesis (kab) cluster
containing an annotated N-prenyltransferase, α-ketoglutarate
(αKG) dependent dioxygenase, and several retrotransposable
After reconstituting DsKabA activity, we next aimed to
determine if DsKabC could directly convert prekainic acid to kainic
acid. Incubation of DsKabC with prekainic acid, αKG, L-ascorbate,
and Fe2+ resulted in the conversion of prekainic acid to two
products with the expected mass of kainic acid (Figure 3).
Addition of the iron chelator EDTA nearly abolished activity. The
major product had the same HPLC retention time and MS of
commercially available kainic acid, and was further confirmed by
to be kainic acid following NMR structural elucidation.
Examination of the in vitro products of three additional algal KabC
elements (integrase, reverse transcriptase, and RNase
H
domains) (Figure 2C). Many organisms, including bacteria, fungi,
plants, and unicellular algae, are known to cluster genes within
the same biosynthetic pathway.[18,21,22] The kab gene clustering
is consistent with early examples of red algae clustering genes[19]
and begins to suggest that it may be a feature in red algal
specialized metabolism as well. To evaluate whether these
features are conserved within other kab clusters, we performed
genome sequencing of a second known kainic acid producer,
Palmaria palmata,[23] to determine the genetic context of its kab
genes. Using one MinION chip and no genome assembly steps,
we were able to identify a single 47 kb read that contained both
ppkabA and ppkabC genes. As with D. simplex, the kainic acid
genes are tightly clustered (Figure 2D). However, the gene
synteny is not conserved with a different gene orientation and a
short 1.1 kb intergenic region between ppkabA and ppkabC that
lacks retrotransposable elements.
Analysis of the NCBI transcriptome database identified
similar kab transcripts from four different red algae, namely
Grateloupia filicina, Palmaria hecatensis, Rhodophysema
elegans, and the previously sequenced P. palmata.[24,25] These
three additional red algae are not known kainic acid producers
and demonstrate that the genes responsible for biosynthesis are
found throughout distinct red algae lineages (Figure S2).
Sequence alignment of the KabA N-prenyltransferase and the
Figure 3. LCMS analysis of DsKabA and DsKabC in vitro assays. Each trace
represents the (M-H)- extracted ion chromatogram for prekainic acid (214.1 
0.5 m/z) and kainic acid (212.1  0.5 m/z). DsKabA converts L-Glu and DMAPP
to prekainic acid in a Mg2+ dependent manner (purple traces). DsKabC can
convert prekainic acid (PKA) to kainic acid (KA) and kainic acid lactone (KA
Lactone), but turnover is nearly undetectable when the iron chelator EDTA is
added (cyan traces).
This article is protected by copyright. All rights reserved.

10.1002/anie.201902910
Angewandte Chemie International Edition
COMMUNICATION
(Figure 4B). We developed a streamlined strategy in which
prekainic acid was synthesized and the entire unpurified crude
reaction mixture was added to the E. coli medium. After 40 h, we
observed the nearly complete consumption of an 8 mmol
prekainic acid synthetic reaction in a 3
X 1 L E. coli
biotransformation to kainic acid (Figure S11). Employing a two-
step purification procedure using activated carbon followed by
preparatory reverse phase HPLC, we purified 1.1 g of kainic acid
with a combined overall isolated yield of 32% and > 95% purity as
assessed by NMR (Figure 4C and S12).
Although there are already many scalable synthetic
processes for kainic acid, the procedures require at least six
synthetic transformations with yields < 40% (Figure 1).[14,16] In
contrast, biology has evolved an extremely efficient two-step
biosynthetic process. The first step, catalyzed by the N-
prenyltransferase KabA, is readily replicated using established
synthetic procedures. In contrast, the second step, in which KabC
catalyzes the stereocontrolled formation of the trisubstituted
pyrrolidine ring, is not easily accomplished synthetically. By
combining the strengths of prekainic acid synthesis and DsKabC-
mediated biocatalysis, we developed a simple and scalable kainic
acid production method that is also economically attractive and
flexible for analog generation.
Figure 4. Discovery of the kainic acid biosynthetic genes enabled two new
kainic acid production routes: (A) one-enzyme chemoenzymatic synthesis and
(B) biotransformation with E. coli cells expressing the dskabC gene on a
pET28 vector. (C) Lyophilized kainic acid in a scintillation vial generated by
biotransformation.
enzymes revealed a similar trend, wherein prekainic acid is
converted to kainic acid and a second compound (Figure S8).
Notably, the KabC ortholog from G. filicinia, GfKabC, produced
primarily this other kainic acid isomer. By scaling up the GfKabC
reaction, we isolated and characterized this product by NMR to
reveal kainic acid lactone (Figure 2B). Kainic acid lactone was
previously isolated from kainic acid producing red algae[26] and
was also found in our D. simplex sample (Figure S7). In contrast
to the agonistic activity of kainic acid, kainic acid lactone has been
shown to be an iGluR antagonist.[27] Curiously, the relative ratio of
kainic acid to kainic acid lactone appears to vary depending on
the particular KabC ortholog (Figure S8). While the biochemical
basis for this differential product distribution is an ongoing focus
of study, we suggest a chemical mechanism analogous to that
proposed for DabC[18] which diverges following pyrrolidine ring
formation to rationalize the observed chemotype (Figure S9).
Once the Kab enzymatic functions were validated, we next
focused on developing a scalable kainic acid production system
that would take advantage of the strengths of both chemical
synthesis and biocatalysis. Instead of purifying recombinant KabA
and isolating its product from a large-scale biochemical reaction,
we readily synthesized prekainic acid by reductive amination of L-
glutamate with 3-methyl-2-butenal (Figure 4). The stereospecific
cyclization reaction to form the kainic acid pyrrolidine ring, on the
other hand, is synthetically challenging and is reminiscent of the
cobalt-mediated radical cyclization strategy employed by Baldwin
and coworkers in the 1990s on an N-prenylated amino acid
substrate (Figure 1C).[17] Rather, we explored using KabC to
produce kainic acid. We first evaluated an in vitro system that
used prekainic acid, αKG, Fe2+, and purified DsKabC from 1 L of
E. coli cells. After a 16 h incubation, 10 mg of synthetic prekainic
acid was completely converted to produce 4.6 mg kainic acid with
a 46% isolated yield and a 26% overall isolated yield over two
steps (Figure 4A and S10). Although this chemoenzymatic route
was successful, the need to purify DsKabC prevents this method
from being applicable for large scale production of kainic acid.
Therefore, we attempted a biotransformation strategy that would
bypass the need to purify enzymes and directly convert synthetic
prekainic acid to kainic acid in E. coli cells expressing DsKabC
Overall, combining genomic information with in vitro
enzymatic characterization enabled us to not only discover the
enzymes responsible for the biosynthesis of a seaweed natural
product, but also to develop new methods to produce this
important research tool.
Experimental Section
Experimental details including DNA sequencing, protein expression,
protein purification, production isolation, chemical synthesis and
spectroscopic characterization are provided in the Supporting Information
online. All sequence data has been deposited to NCBI databases. The
Digenea simplex and Palmaria palmata bioprojects have been deposited
as PRJNA509898 and PRJNA509900, respectively. A complete list of all
accession numbers can be found in Supplementary Table 3.
Acknowledgements
We thank T. Teruya (University of Ryukyus) for collecting and
sending us Digenea simplex, G. Saunders (University of New
Brunswick) for sending us DNA and algal samples, T. Steele and
E. Ricciardelli (University of California at San Diego) for technical
support, and G. Rouse (Scripps Institution of Oceanography) and
S. Matsunaga (University of Tokyo) for helpful discussions. This
work was supported by the US National Institutes of Health (R01-
GM085770 to B.S.M.), the Simons Foundation Fellowship of the
Life Science Research Foundation to J.R.C., the Natural
Sciences and Engineering Research Council of Canada (NSERC-
PDF to S.M.K.M.), and the American Society for Pharmacognosy
undergraduate research award to M.L.M.
This article is protected by copyright. All rights reserved.

10.1002/anie.201902910
Angewandte Chemie International Edition
COMMUNICATION
[15]
[16]
[17]
[18]
S. Takita, S. Yokoshima, T. Fukuyama, Org. Lett. 2011, 13, 2068–
70.
Conflict of Interest
M. Zhang, K. Watanabe, M. Tsukamoto, R. Shibuya, H. Morimoto,
T. Ohshima, Chem. - A Eur. J. 2015, 21, 3937–41.
J. E. Baldwin, M. G. Moloney, A. F. Parsons, Tetrahedron 1990, 46,
7263–82.
J.R.C., S.M.K.M, and B.S.M. have submitted a provisional patent
on the production of kainic acid using the described methods.
Keywords: kainic acid
•
biosynthesis
•
red algae
•
J. K. Brunson, S. M. K. McKinnie, J. R. Chekan, J. P. McCrow, Z. D.
Miles, E. M. Bertrand, V. A. Bielinski, H. Luhavaya, M. Oborník, G.
J. Smith, et al., Science 2018, 361, 1356–8.
biotransformation • natural product
[1]
[2]
[3]
T. Higa, M. Kuniyoshi, J. Toxicol. - Toxin Rev. 2000, 19, 119–37.
I. Nitta, H. Watase, Y. Tomiie, Nature 1958, 181, 761–2.
S. Murakami, T. Takemoto, Z. Shimizu, Yakugaku Zasshi 1953, 73,
1026–8.
[19]
[20]
S. H. Brawley, N. A. Blouin, E. Ficko-Blean, G. L. Wheeler, M. Lohr,
H. V. Goodson, J. W. Jenkins, C. E. Blaby-Haas, K. E. Helliwell, C.
X. Chan, et al., Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E6361-70.
T. P. Michael, F. Jupe, F. Bemm, S. T. Motley, J. P. Sandoval, C.
Lanz, O. Loudet, D. Weigel, J. R. Ecker, Nat. Commun. 2018, 9,
541.
[4]
[5]
Y. Komiya, A. Kobayashi, Jpn. J. Med. Sci. Biol. 1965, 18, 1–17.
S. H. Lee, S. C. Kang, J. H. Ahn, J. W. Lee, H. J. Rim, Korean J.
Parasitol. 1972, 10, 79–85.
[21]
[22]
S. Boycheva, L. Daviet, J. Wolfender, T. B. Fitzpatrick, Trends Plant
Sci. 2014, 19, 447–59.
[6]
[7]
J.-F. Tremblay, Chem. Eng. News 2000, 78, 14–5.
P. Werner, M. Voigt, K. Keinänen, W. Wisden, P. H. Seeburg,
Nature 1991, 351, 742–4.
P. Schläpfer, P. Zhang, C. Wang, T. Kim, M. Banf, L. Chae, K.
Dreher, A. K. Chavali, R. Nilo-Poyanco, T. Bernard, et al., Plant
Physiol. 2017, 173, 2041–59.
[8]
R. Dingledine, K. Borges, D. Bowie, S. F. Traynelis, Pharmacol.
Rev. 1999, 51, 7–61.
[23]
[24]
U. P. Ramsey, C. J. Bird, P. F. Shacklock, M. V. Laycock, J. L. C.
Wright, Nat. Toxins 1994, 2, 286–92.
[9]
D. R. Hampson, J. L. Manalo, Nat. Toxins 1998, 6, 153–8.
D. Lodge, Neuropharmacology 2009, 56, 6–21.
X. Y. Zheng, H. L. Zhang, Q. Luo, J. Zhu, J. Biomed. Biotechnol.
2011, 2011, 457079.
[10]
[11]
N. Matasci, L. Hung, Z. Yan, E. J. Carpenter, N. J. Wickett, S.
Mirarab, N. Nguyen, T. Warnow, S. Ayyampalayam, M. Barker, et
al., Gigascience 2014, 3, 17.
[12]
[13]
[14]
Y. Ben-Ari, J. Lagowska, E. Tremblay, G. Le Gal La Salle, Brain
Res. 1979, 163, 176–9.
[25]
[26]
[27]
G. W. Saunders, C. Jackson, E. D. Salomaki, Mol. Phylogenet.
Evol. 2018, 119, 151–9.
M. Lévesque, M. Avoli, Neurosci. Biobehav. Rev. 2013, 37, 2887–
99.
M. Miyasaki, H. Watanabe, T. Takano, A. Morimoto, Yakugaku
Zasshi 1956, 76, 189–91.
C. I. Stathakis, E. G. Yioti, J. K. Gallos, European J. Org. Chem.
2012, 2012, 4661–73.
O. Goldberg, A. Luini, V. I. Teichberg, Neurosci. Lett. 1981, 23,
187–91.
This article is protected by copyright. All rights reserved.

10.1002/anie.201902910
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Jonathan R. Chekan, Shaun M. K.
McKinnie, Malia L. Moore, Shane G.
Poplawski, Todd P. Michael, & Bradley
S. Moore*
Page No. – Page No.
Scalable biosynthesis of the seaweed
neurochemical kainic acid
Kainic acid is a marine natural product that has been used as an anthelmintic agent
for centuries and, more recently, a neurological tool. Using a genomic sequencing
approach, the genes responsible for the biosynthesis of kainic acid were discovered
in two different seaweeds. In vitro characterization validated enzymatic activity and
enabled gram scale production of kainic acid using a biotransformation approach.
This article is protected by copyright. All rights reserved.