Chapter 1
Introduction

Rainer Kurmayer1*, Kaarina Sivonen2 and Nico Salmaso3

1Research Institute for Limnology, University of Innsbruck, Mondsee, Austria

2Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Helsinki, Finland

3Research and Innovation Centre, Fondazione Edmund Mach - Istituto Agrario di S. Michele all'Adige, S. Michele all'Adige, Italy

* Corresponding author: rainer.kurmayer@uibk.ac.at

1.1 A Brief Historical Overview

During the last two decades, genetic methods have significantly increased our understanding of the distribution of genes involved in the production of toxins within the phylum of cyanobacteria (e.g. Sivonen and Börner, 2008; Dittmann et al., 2013; Méjean and Ploux, 2013). Early on the synthesis pathways of microcystin in the three genera Microcystis, Planktothrix, and Anabaena (Tillett et al., 2000; Christiansen et al., 2003; Rouhiainen et al., 2004) and of the closely related nodularin have been elucidated (Moffitt and Neilan, 2004). Further, the elucidation of the genes involved in cyanotoxin synthesis increased the understanding of its inheritance and evolution, (e.g. the phylogenetically derived conclusion on the evolutionary age of the microcystin/nodularin synthesis pathway) (Rantala et al., 2004) implying that potentially all cyanobacteria are able to produce microcystins, and, indeed, the number of cyanobacterial genera discovered to produce microcystins is consistently increasing (e.g. Calteau et al., 2014).

Subsequently, the elucidation of the synthesis pathways of other toxins has been achieved, that is first results suggested the involvement of polyketide synthases (PKS) and an amidinotransferase in the synthesis of cylindrospermopsin in Aphanizomenon (Shalev-Alon et al., 2002; Kellmann et al., 2006) which then led to the identification of the first putative cylindrospermopsin gene cluster (cyr) in Cylindrospermopsis (Mihali et al., 2008). Other cylindrospermopsin synthesis gene clusters followed, in particular for Oscillatoria (Mazmouz et al., 2010), for Aphanizomenon (Stüken and Jakobsen, 2010), Raphidiopsis curvata, and Cylindrospermopsis raciborskii (Jiang et al., 2014). In general, compared with mcy genes (encoding the synthesis of microcystins), there is more shuffling of genes, and eleven genes cyrA-K are thought to make the core of the cyr gene cluster.

Similarly, candidate genes for saxitoxin biosynthesis have been isolated and the sequence of the complete putative saxitoxin biosynthetic gene cluster (sxt) was obtained (Kellmann et al., 2008a,b). This work started with screening of putative saxitoxin biosynthetic enzymes in cyanobacterial isolates, using a degenerate PCR approach, resulting in identification of an O-carbamoyltransferase that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in paralytic shellfish poisoning (PSP) strains of cyanobacteria and had a high sequence similarity to each other (Kellmann et al., 2008a). The first sxt gene cluster was sequenced from Cylindrospermopsis, and orthologous gene clusters from Anabaena, Aphanizomenon, Raphidiopsis, and Lyngbya followed (Murray et al., 2011). Genetic proof (e.g. by experimental gene inactivation) for the role of this gene cluster in saxitoxin biosynthesis is lacking. However, in the absence of suitable tools of genetic transformation, the functions of the ORF (open reading frame) were bioinformatically inferred, and this prediction was combined with the liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates (Kellmann and Neilan, 2007; Kellmann et al., 2008b).

The first anatoxin-a synthesis gene cluster (ana) was sequenced from Oscillatoria (Méjean et al., 2009). Subsequently anatoxin-a gene clusters were described from Anabaena (Rantala-Ylinen et al., 2011) and Cylindrospermum (Calteau et al., 2014). In the following, the genetic basis of microcystin/nodularin, cylindrospermopsin, saxitoxin, and anatoxin synthesis is described in more detail.

1.2 The Genetic Basis of Toxin Production

1.2.1 Microcystin and Nodularin

Microcystins are produced by planktonic freshwater genera Microcystis, Planktothrix, Dolichospermum, Nostoc, and Fischerella (Dittmann et al., 2013). Early studies, however, also documented microcystin production in a broader range of terrestrial genera, for example in Hapalosiphon (Prinsep et al., 1992) and later in Nostoc symbionts associated with fungi (Oksanen et al., 2004). In addition numerous freshwater and brackish water genera (e.g. Arthrospira, Oscillatoria, Phormidium, Pseudanabaena, Synechococcus, Synechocystis) have been reported to produce microcystins (Sivonen and Börner, 2008; Fiore et al., 2009; Bernard et al., 2017). In contrast, the closely related nodularin has been characterized from the brackish water species Nodularia spumigena and Nostoc (Bernard et al., 2017), while in the marine sponge, Theonella swinhoei, a nodularin analogue called motuporin has been found (de Silva et al., 1992). The sponge is known to harbor cyanobacterial symbionts. Microcystins are known for their toxicity because of the inhibition of eukaryotic protein phosphatases 1 and 2A resulting in the hyperphosphorylation and breakdown of the structural protein skeleton (Carmichael, 1994). Not at least because of the interference with eukaryotic signaling cascades, microcystins are considered tumor promotors under sublethal exposure conditions (Zhou et al., 2002).

Microcystins are cyclic heptapeptides and share the common structure cyclo (- D-Ala(1) - X(2) - D-MAsp(3) - Z(4) - Adda(5) - D-Glu(6) - Mdha(7)), where X and Z are variable L-amino acids (e.g., microcystin (MC)-LR refers to leucine and arginine in the variable positions), D-MAsp is D-erythro-ß-iso-aspartic acid, Adda is (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and Mdha is N-methyl-dehydroalanine (Carmichael et al., 1988). Considerable structural variation has been reported, most frequently in positions 2, 4, and 7 of the molecule, and a large number of structural variants have been characterized (molecular weight 909 - 1115 Da), either from field samples or from isolated strains (e.g. Diehnelt et al., 2006; Spoof and Catherine, 2017). Nodularin (824 Da) and motuporin (812 Da) are both pentapeptides containing N-methyl-dehydrobutyrine (Mdhb) instead of Mdha(7) and lack D-Ala(1) and X(2) when compared with microcystin. Nodularin differs from motuporin due to the substitution of L-Arg(4) by L-Val(4) (Fig. 1.1).

Figure 1.1 Scheme of the genetic basis of microcystin/nodularin synthesis in sequenced cyanobacterial genera. Arrows mark the bi-directional promotor region (from Tillett et al., 2000; Christiansen et al., 2003; Moffitt and Neilan, 2004; Rouhiainen et al., 2004; Fewer et al., 2013; Shih et al., 2013).

The biosynthesis of microcystin is catalyzed by nonribosomal peptide synthesis (NRPS) via the thio-template mechanism. This biosynthetic pathway has been intensively investigated in different bacteria and fungi, as their end products are often of great pharmaceutical value (Fischbach and Walsh, 2006). At present six gene clusters from five genera (Microcystis, Planktothrix, Anabaena, Nodularia, Fischerella) responsible for the biosynthesis of microcystin have been sequenced (Tillett et al., 2000; Christiansen et al., 2003; Rouhiainen et al., 2004; Moffitt and Neilan, 2004; Fewer et al., 2013; Shi et al., 2013) and the involvement in the production of microcystins could be proven by genetic manipulation in Microcystis and Planktothrix (Dittmann et al., 1997; Christiansen et al., 2003). The whole mcy gene cluster comprises a minimum of nine genes (ca. 55 kb) consisting of PKS, nonribosomal peptide synthetases (NRPS), and tailoring enzymes. It has a modular structure (Fig. 1.1), each module containing specific functional domains for activation (aminoacyl adenylation (A)-domains), thioesterification (thiolation domains) of the amino acid substrate and for the elongation (condensation (C)-domains) of the growing peptide. McyD, McyE, and McyG are responsible for the production of the amino acid Adda and the activation and condensation of D-glutamate. McyA, McyB, and McyC are NRPS and responsible for the incorporation of the other five amino acids in positions 7, 1, 2, 3, and 4 of the molecule (Tillett et al., 2000). Synthesis is thought to start with activation of phenyllactate through the adenylation domain of McyG (Hicks et al., 2006) followed by extension of the polyketide through McyD and McyE. The polyketide is then condensed with D-glutamate through McyE forming the core of the microcystin peptide (comprising the Adda side chain and the Glutamate). The residual amino acids are then condensed through McyA, B, C proteins and finally the peptide is cyclized through a dedicated type I thioesterase (Tillett et...