Genome driven discovery of new natural products

Much of the natural product chemical diversity observed in nature is attributed to versatile non-ribosomal peptide synthase (NRPS) and polyketides synthetase (PKS) biosynthetic pathways. We have shown that cyanobacterial genomes encode a huge diversity of these biosynthetic pathways (Shih et al. 2013, Wang et al. 2011, 2012, 2014, Shih et al. 2013). Likewise, ribosomal gene clusters, recently shown to produce complex peptides through the post-translational modification of short precursor proteins, are very common in cyanobacterial genomes (Leikoski et al. 2010, 2013). However, the end-products of the vast majority of these pathways are currently unknown (Calteau et al. 2015, Dittmann et al. 2015). Our genome mining studies at the phylum level have demonstrated the unexpected widespread distribution and biological diversity of secondary metabolite biosynthetic gene clusters (Shih et al. 2013, Wang et al. 2014, Calteau et al. 2014).

We carry out genome driven discovery of new bioactive peptides from cyanobacteria (Wang et al. 2014). Through the use of genome sequences we discovered new families of protease inhibitors and antifungal peptides, new enzymatic machinery for making cyclic peptides (Vestola et al. 2015, Shishido et al. 2015). However, the lack of genetic system and slow growth times of cyanobacteria and most other bioactive compound producing organisms is a bottleneck in natural product discovery and slows the pace at which research can be carried out (Dittmann et al. 2015). We are developing methods to overcome these limitations and accelerate the discovery of new natural products from cyanobacteria. We are also interested in understanding how cyanobacteria make natural products and in understanding how the enzymatic machinery for producing peptides functions. Biochemical investigations shed light on how these enzymes work together to create cyclic peptides. We use phylogenetic analyses of the biosynthetic machinery to show how natural products evolve in nature and this ultimately may provide clues to how cyanobacteria can be rewired to make new natural products (Calteau et al. 2014, Dittmann et al. 2015).

Nonribosomal peptides

Several cyclic, branched, or linear bioactive peptides of bacteria and lower eukaryotes are produced non-ribosomally by multidomain peptide synthetases, employing a thiotemplate mechanism. Different domains of peptide synthetases act as independent enzymes whose function is to join one amino acid to the growing polypeptide chain and make possible modifications. The specific order of the domains forms the protein template that defines the sequence of the incorporated amino acids. Our genome mining studies conducted at the phylum and domain level have demonstrated that peptide synthetase genes are common in cyanobacteria (Wang et al. 2014, Calteau et al. 2014, Dittmann et al. 2015). One hepatotoxic strain, Anabaena 90, isolated from a Finnish lake, Vesijärvi, was selected as a model strain to study the peptide synthetase system. Three classes of cyclic peptides have been isolated and characterized from Anabaena sp. 90: two types of heptapeptides, microcystins and anabaenopeptilides, and one type of hexapeptides, anabaenopeptins. The microcystin, anabaenopeptilide, anabaenopeptin, hassallidin and anabaenolysin synthetase gene clusters have been characterized from Anabaena sp. 90 or other Anabaena strains in our group (Rouhiainen et al. 2000, 2004, 2010, Vestola et al. 2014, Shishido et al. 2015). Peptide synthetase gene clusters from Nostoc sp. 152 (Fewer et al. 2011, 2013) and Nodularia spumigena CCY9414 have also been characterized (Fewer et al. 2009, 2013, Liu et al. 2015). We have also studied non-ribosomal peptides with cytotoxic and antimicrobial effects from other cyanobacteria. Information about genes involved in the synthesis of these compounds, occurrence of these genes and production of these compounds are investigated. Through phylogenetic analysis it is possible to analyze the distribution of the genes among distant or close related cyanobacterial strains (Wang et al. 2014, Calteau et al. 2014). Eventually, bioassays trying to solve the ecological role or analysis of mechanism of action of these compounds will be performed.

Ribosomal peptides

Cyanobactins are small cyclic peptides recently described from cyanobacteria (Sivonen et al. 2010). They are formed through the proteolytic cleavage and post-translational modification of short precursor proteins and exhibit anti-tumor, cytotoxic or multi-drug reversing activities. Novel cyanobactins, anacyclamides, piricyclamides and linear aeruginosamide and viridisamide were recently found to be common in various cyanobacteria (Leikoski et al. 2010, 2012, 2013). Our future research will focus on biosynthesis and detection of new cyanobactins in cyanobacteria through genome mining. Bacteriocin gene cluster were found to be common in cyanobacteria (Wang et al. 2011, Shih et al. 2013). Our work has shown that ribosomal peptide biosynthetic gene clusters are very common in cyanobacteria (Wang et al. 2011, Shih et al. 2013). However, the vast majority of these biosynthetic pathways have no known end product associated with them. Work is now underway to unravel the complex distribution of ribosomal peptides in cyanobacteria and develop novel methods to express silent gene clusters in heterologous hosts. This work has led to the discovery of novel peptide with unusual posttranslational modifications and antimicrobial bioactivities.

Whole genome Sequencing

We have sequenced a number of model toxin producing cyanobacteria from Finnish lakes and the Baltic Sea from our culture collection. We have sequenced the complete genome of microcystin producing Anabaena sp. 90 in collaboration with the Beijing Genomics Institute, China (Wang et al. 2012). This strain was isolated from Lake Vesijärvi and produces microcystins. The whole genome of anatoxin-a producing Anabaena strain 37 was initiated in collaboration with Institute of Biomedical Technologies, Italy. The akinete forming Anabaena strain ITU33S10, anabaenolysin producing Anabaena strain XSPORK 2A, XPORK13A andXPORK15F are currently being sequenced. These genome projects will provide further insights into the proliferation of cyanobacteria in Finnish lakes and the production of bioactive compounds by cyanobacteria. We were also involved in the genome project of Nodularia spumigena CCY9414 which was isolated from near Bornholm in the Baltic Sea and produces the hepatotoxin nodularin (Voss et al. 2013). The genome project was initiated by Lucas Stal at NIOO in Holland and sequenced at the Craig Venter Institute in the US as part of the Moore Foundations Marine Microbiology initiative. We have now obtained genome sequences for 60 further strains from benthic and planktonic environments of the Baltic Sea and Finnish lakes. Analysis of these genome sequences have provided new insights into the ecology of these organisms, how they are adapted to their environment and the types of bioactive compounds they produce (Wang et al. 2012, Voss et al. 2013, Leikoski et al. 2013, Calteau et al. 2014, Vestola et al. 2014, Shishido et al. 2015).