222 Marine Macro- and Microalgae: An Overview
Toxin production approaches
Dinoflagellate-generated toxins unfold a wide applicability in the health sector; however, most such
molecules have not progressed beyond discovery stages because of limited availability from natural
sources—and almost insurmountable difficulty to obtain via chemical synthesis, owing to their structural
complexity. Laboratory cultures of dinoflagellates have also proven hard to establish, or failed to yield
the intended compounds (Gallardo-Rodríguez et al. 2012a).
Lack of sufficient quantities has systematically hampered further biochemical investigation and
clinical testing, thus compromising eventual development into commercial products. Very few commercial
biotoxins are available for purchase, and the existing ones reach outrageous prices—ranging from 1,000
to 500,000 € per milligram. This is the case of commercial okadaic acid produced from Prorocentrum
spp., a cytotoxic inhibitor of protein phosphatase-2A, able to alter the phosphorylation state of cellular
proteins, thus leading to collapse of normal regulatory pathways—for example, induced downregulation
of T-cell receptor expression compromising T-cell activation (Valdiglesias et al. 2013). Another example
is azaspiracid, a polyether characterized by a cyclic-amine or aza group, produced by Azadinium spp.
(Tillmann et al. 2009); azaspiracid functions as an activator of c-Jun-N-terminal kinase and caspases
(implicated in stress signaling pathways) (Cao et al. 2010), but may also interfere with gene expression
and inhibit cell cholesterol levels—especially in T-lymphocytes (Twiner et al. 2008). Azaspiracid-3—an
analogue of this bioactive compound, can reach prices of 500 to 600 € just for 1 µg of product. In any
case, Care should be taken when these type of commercial substances are purchased—as their producton
may be easily discontinued, and the purity/quantity allegedly claimed by companies may be questioned
(Quilliam 2003).
Different strategies have been applied to enhance production of target dinoflagellate derived-toxins.
Nevertheless, some of those have shown to be complex, raising several challenges ahead.
Chemical synthesis and genetic engineering
The potential of dinoflagellate biotoxins and derivatives has been severely constrained by inability
to attain acceptable productivities, sufficient to respond to the increase in demand for investigational
purposes (i.e., for assessment of pharmacological potential and activity), as well as for preclinical studies
and clinical trials. Strategies including chemical synthesis of most dinoflagellate toxins are theoretically
possible, but still excessively expensive (except okadaic acid)—while most of them comprise several
laborious and intrincate steps (more than 100 steps may actually be required); more practical synthetic
routes remain a challenge, unlikely to succeed in the short run. However, it is important to highlight
that this approach has allowed a few advances concerning elucidation of structure and mode of action
of some complex metabolites derived from dinoflagellates [e.g., brevetoxin A (Gawley et al. 1995),
gambierol (Fuwa et al. 2002), gymnocin A (Tsukano and Sasaki 2006), and azaspiracid-1 (Nicolaou et
al. 2006)]. On the other hand, genetic improvements and metabolic engineering are very hard, since the
genome of dinoflagellates is complex (Jaeckisch et al. 2011)—and available genetic tools have chiefly
been developed for nondinoflagellate microalgae and target biofuel production (Radakovits et al. 2010).
Metabolic engineering is not easy in dinoflagellate cells, as they present astonishingly large and
complex genomes—with a great many introns, bearing redundant repetitive noncoding sequences
(McEwan et al. 2008) and a high proportion of unusual bases with a fifth base replacing uracyl
in their DNA (Wisecaver and Hackett 2011). DNA content makes it difficult to perform simple
genomic hybridization, like Southern blots, and it is impractical to construct genomic libraries or to
consider sequencing their genome in the first place. As dinoflagellates are lacking a general
transformation system and experience difficulties to grow on solid media, cloning will prove a very
complicated procedure. Genetic transformation was only reported in two different dinoflagellates
(Symbiodinium sp. and Amphidinium sp.) (ten Lohuis and Miller 1998), and no further attempts have
meanwhile been reported. Furthermore, dinoflagellate genes lack recognizable promoter features and
common eukaryotic transcription factor binding sites (Jaeckisch et al. 2011), and their toxin production
Toxin production approaches
Dinoflagellate-generated toxins unfold a wide applicability in the health sector; however, most such
molecules have not progressed beyond discovery stages because of limited availability from natural
sources—and almost insurmountable difficulty to obtain via chemical synthesis, owing to their structural
complexity. Laboratory cultures of dinoflagellates have also proven hard to establish, or failed to yield
the intended compounds (Gallardo-Rodríguez et al. 2012a).
Lack of sufficient quantities has systematically hampered further biochemical investigation and
clinical testing, thus compromising eventual development into commercial products. Very few commercial
biotoxins are available for purchase, and the existing ones reach outrageous prices—ranging from 1,000
to 500,000 € per milligram. This is the case of commercial okadaic acid produced from Prorocentrum
spp., a cytotoxic inhibitor of protein phosphatase-2A, able to alter the phosphorylation state of cellular
proteins, thus leading to collapse of normal regulatory pathways—for example, induced downregulation
of T-cell receptor expression compromising T-cell activation (Valdiglesias et al. 2013). Another example
is azaspiracid, a polyether characterized by a cyclic-amine or aza group, produced by Azadinium spp.
(Tillmann et al. 2009); azaspiracid functions as an activator of c-Jun-N-terminal kinase and caspases
(implicated in stress signaling pathways) (Cao et al. 2010), but may also interfere with gene expression
and inhibit cell cholesterol levels—especially in T-lymphocytes (Twiner et al. 2008). Azaspiracid-3—an
analogue of this bioactive compound, can reach prices of 500 to 600 € just for 1 µg of product. In any
case, Care should be taken when these type of commercial substances are purchased—as their producton
may be easily discontinued, and the purity/quantity allegedly claimed by companies may be questioned
(Quilliam 2003).
Different strategies have been applied to enhance production of target dinoflagellate derived-toxins.
Nevertheless, some of those have shown to be complex, raising several challenges ahead.
Chemical synthesis and genetic engineering
The potential of dinoflagellate biotoxins and derivatives has been severely constrained by inability
to attain acceptable productivities, sufficient to respond to the increase in demand for investigational
purposes (i.e., for assessment of pharmacological potential and activity), as well as for preclinical studies
and clinical trials. Strategies including chemical synthesis of most dinoflagellate toxins are theoretically
possible, but still excessively expensive (except okadaic acid)—while most of them comprise several
laborious and intrincate steps (more than 100 steps may actually be required); more practical synthetic
routes remain a challenge, unlikely to succeed in the short run. However, it is important to highlight
that this approach has allowed a few advances concerning elucidation of structure and mode of action
of some complex metabolites derived from dinoflagellates [e.g., brevetoxin A (Gawley et al. 1995),
gambierol (Fuwa et al. 2002), gymnocin A (Tsukano and Sasaki 2006), and azaspiracid-1 (Nicolaou et
al. 2006)]. On the other hand, genetic improvements and metabolic engineering are very hard, since the
genome of dinoflagellates is complex (Jaeckisch et al. 2011)—and available genetic tools have chiefly
been developed for nondinoflagellate microalgae and target biofuel production (Radakovits et al. 2010).
Metabolic engineering is not easy in dinoflagellate cells, as they present astonishingly large and
complex genomes—with a great many introns, bearing redundant repetitive noncoding sequences
(McEwan et al. 2008) and a high proportion of unusual bases with a fifth base replacing uracyl
in their DNA (Wisecaver and Hackett 2011). DNA content makes it difficult to perform simple
genomic hybridization, like Southern blots, and it is impractical to construct genomic libraries or to
consider sequencing their genome in the first place. As dinoflagellates are lacking a general
transformation system and experience difficulties to grow on solid media, cloning will prove a very
complicated procedure. Genetic transformation was only reported in two different dinoflagellates
(Symbiodinium sp. and Amphidinium sp.) (ten Lohuis and Miller 1998), and no further attempts have
meanwhile been reported. Furthermore, dinoflagellate genes lack recognizable promoter features and
common eukaryotic transcription factor binding sites (Jaeckisch et al. 2011), and their toxin production