Antifungal and Antileukemic Compounds from Cyanobacteria: Bioactivity, Biosynthesis, and Mechanism of Action

ANTIFUNGAL AND ANTILEUKEMIC COMPOUNDS FROM CYANOBACTERIA: BIOACTIVITY, BIOSYNTHESIS, AND MECHANISM OF ACTION

Anu Humisto

Department of Microbiology Faculty of Agriculture and Forestry University of Helsinki, Finland

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Auditorium I at Info Centre Korona, Viikinkaari 11, on 23rd November 2018, at 12 o'clock noon.

Helsinki 2018 Supervisors Professor Kaarina Sivonen Department of Microbiology University of Helsinki, Finland Professor Lars Herfindal Department of Clinical Science University of Bergen, Norway Docent Jouni Jokela Department of Microbiology University of Helsinki, Finland

Reviewers Docent Päivi Tammela Division of Pharmaceutical Biosciences Faculty of Pharmacy University of Helsinki, Finland Professor Lena Gerwick Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego, USA

Thesis evaluation committee Professor Per Saris Department of Microbiology University of Helsinki, Finland Academy Fellow Miia Mäkelä Department of Microbiology University of Helsinki, Finland

Opponent Professor Jeanette H. Andersen Norwegian College of Fishery Science University of Tromsø, Norway

Custos Professor Kaarina Sivonen Department of Microbiology University of Helsinki, Finland

Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae Cover: Lake Löytänejärvi (Pori, Finland), light micrograph of Nostoc sp. UHCC 0450, and transmission electron micrograph of MOLM-13 cell organelles. All figures by Anu Humisto.

ISSN 2342-5423 (print) ISSN 2342-5431 (online) ISBN 978-951-51-4658-8 (paperback) ISBN 978-951-51-4659-5 (PDF) http://ethesis.helsinki.fi Unigrafia Helsinki 2018 Table of contents

LIST OF ORIGINAL PUBLICATIONS ...... III

LIST OF RELATED PUBLICATIONS ...... IV

ABBREVIATIONS ...... V

ABSTRACT ...... VI

TIIVISTELMÄ ...... VII

1 INTRODUCTION ...... 1

1.1 NATURAL PRODUCTS AND THEIR IMPORTANCE ...... 1 1.2 CYANOBACTERIA AND THEIR NATURAL PRODUCTS ...... 2 1.3 BIOACTIVITY ...... 4 1.3.1 Anticancer activity ...... 5 1.3.2 Antifungal activity ...... 9 1.4 BIOSYNTHESIS ...... 16 1.4.1 Polyketide synthesis ...... 17 1.4.2 Nonribosomal peptide synthesis and hybrid synthesis ...... 18 1.5 MECHANISM OF ACTION ...... 19

2 STUDY AIMS ...... 22

3 SUMMARY OF MATERIALS AND METHODS ...... 23

3.1 STRAINS AND CELL LINES USED IN THIS THESIS ...... 23 3.2 SUMMARY OF METHODS ...... 23

4 SUMMARY OF RESULTS AND DISCUSSION ...... 25

4.1 BIOACTIVE CYANOBACTERIAL COMPOUNDS ...... 25 4.1.1 Antileukemic activity (I) ...... 25 4.1.2 Antifungal or cytotoxic activity (II, IV) ...... 26 4.1.3 Culture collections and screening (I–IV) ...... 29 4.2 DISCOVERING BIOSYNTHESIS PATHWAYS (III) ...... 30 4.3 ELUCIDATING MECHANISM OF ACTION (IV) ...... 3 5

5 CONCLUSIONS AND FUTURE PROSPECTS ...... 39

6 ACKNOWLEDGEMENTS ...... 41

7 REFERENCES ...... 42

I II List of original publications

This thesis is based on the following publications:

I Humisto A, Herfindal L, Jokela J, Karkman A, Bjørnstad R, Choudhury RR, Sivonen K. 2016. Cyanobacteria as a source for novel anti-leukemic compounds. Current Pharmaceutical Biotechnology, 17(1):78–91.

II Shishido TK, Humisto A, Jokela J, Liu L, Wahlsten M, Tamrakar A, Fewer DP, Permi P, Andreote APD, Fiore MF, Sivonen K. 2015. Antifungal compounds from cyanobacteria. Marine Drugs, 13(4):2124–2140.

III Humisto A, Jokela J, Liu L, Wahlsten M, Wang H, Permi P, Machado JP, Antunes A, Fewer DP, Sivonen K. 2018. The swinholide biosynthesis gene cluster from a terrestrial cyanobacterium, Nostoc sp. strain UHCC 0450. Applied and Environmental Microbiology, 84(3):e02321-17.

IV Humisto A, Jokela J, Teigen K, Wahlsten M, Permi P, Sivonen K, Herfindal L. 2018. Characterization of the membrane interaction of the antifungal and cytotoxic cyclic glycolipopeptide hassallidin. Submitted manuscript.

The publications are referred to in the text by their roman numerals.

Author's contribution

I Anu Humisto participated in the design of the study, performed the experiments, participated in the data analysis, and wrote the first article draft.

II Anu Humisto participated in the design of the study, performed part of the experiments, and participated in the writing of the manuscript.

III Anu Humisto participated in the design of the study, performed the experiments, participated in the data analysis, and wrote the first article draft.

IV Anu Humisto participated in the design of the study, performed the experiments, participated in the data analysis, and wrote the first article draft.

III List of related publications

V Pancrace C, Jokela J, Sassoon N, Ganneau C, Desnos-Ollivier M, Wahlsten M, Humisto A, Calteau A, Bay S, Fewer DP, Sivonen K, Gugger M. 2017. Rearranged biosynthetic gene cluster and synthesis of hassallidin E in Planktothrix serta PCC 8927. ACS Chemical Biology, 12(7):1796–1804.

IV Abbreviations

A Adenylation domain ACP Acyl carrier protein AML Acute myeloid leukemia AT Acyltransferase domain C Condensation domain cf. sp. Uncertainly defined species DH Dehydratase E Epimerization domain

EC50 Half-maximal effective concentration ER Enoyl reductase HGT Horizontal gene transfer

IC50 Half-maximal inhibitory concentration IPC-81 Rat acute myeloid leukemia cell line kb Kilo base pairs KS Ketosynthase KS0 Nonelongating ketosynthase KR Ketoreductase LC-MS Liquid chromatography mass spectrometry Mb Mega base pairs MIC Minimum inhibitory concentration MOA Mechanism of action MOLM-13 Human acute myeloid leukemia cell line MT Methyl transferase NMR Nuclear magnetic resonance NRK Normal rat kidney epithelial cell line NRPS Nonribosomal peptide synthetase OMT O-methyl transferase ORF Open reading frame OX Monooxygenase domain PCP Peptidyl carrier protein PKS Polyketide synthase PS Pyran synthase scp Scytophycin gene cluster sp. Species swi Swinholide gene cluster TE Thioesterase domain UHCC University of Helsinki Cyanobacteria Culture Collection

V Abstract

Nature is a treasury of bioactive natural products that are developed into pharmaceuticals, cosmetics and other industrial applications. Natural products have an impact on all of us, for instance, in the form of antibiotics. Most of the drugs sold today are natural products or their derivatives. However, the need for new natural products has not decreased but is rather increasing. The incidences of certain diseases such as cancers are rising and resistance to treatment is a major problem. Especially prokaryotes and plants produce these intriguing natural products, which display several different bioactivities. Cyanobacteria are photosynthetic prokaryotes that belong to the most prolific sources of bioactive compounds. This study expands the knowledge of cyanobacterial natural products, including their activities, biosynthesis, and mechanisms of action. The University of Helsinki Cyanobacteria Culture Collection was utilized in this study. Cultured cyanobacteria were screened for antileukemic and antifungal activity using cell assays and disk diffusion analyses. Bioactive compounds were identified with spectrometric methods. The screenings revealed several bioactive hits, including antifungal, antileukemic and cytotoxic activities. Novel compound candidates and known compounds from new habitats or genera were found. In addition, novel variants of known compounds were identified. The results from screening were used to select cyanobacterial strains for whole genome sequencing. Genomic analysis was used to identify the biosynthesis gene clusters of the cytotoxic compounds swinholide and scytophycin. In addition, the production of swinholide was confirmed for the first time using axenic cyanobacteria. In the last part of the study, the mechanism of action of hassallidin was determined using cellular assays, imaging, and lipid models (in vitro and in silico). Hassallidin D purified from Anabaena sp. showed cholesterol-dependent lytic activity against eukaryotic cells. The development of a natural product into a drug or other application is a long process, but natural products are needed also in the future. This thesis contributes to this effort by increasing the understanding of several bioactive cyanobacterial natural compounds at both the functional and molecular level.

VI Tiivistelmä

Luonto on kuin aarreaitta, josta ammennamme uusia bioaktiivisia yhdisteitä lääkkeiden, kosmetiikan ja muun teollisuuden käyttöön. Luonnon bioaktiivisilla yhdisteillä on merkit- tävä vaikutus meidän kaikkien elämään, esimerkiksi antibioottien muodossa. Suurin osa myydyistä lääkkeistä onkin luonnonyhdisteitä tai niitä mukailevia synteettisiä yhdisteitä. Uusien luonnonyhdisteiden löytäminen on kuitenkin edelleen tärkeää, sillä joidenkin sai- rauksien on todettu lisääntyneen. Esimerkiksi syöpien määrä on kasvussa, ja lisäksi lääke- resistenssi on vakava ja kasvava ongelma. Erityisesti prokaryootit ja kasvit tuottavat yhdis- teitä, joilla on useita erilaisia bioaktiivisuuksia ja potentiaalia muun muassa lääkeaineiksi. Fotosynteettiset prokaryootit syanobakteerit kuuluvat parhaimpiin bioaktiivisten aineiden tuottajiin. Tässä väitöskirjatutkimuksessa tutkittiin syanobakteerien bioaktiivisia yhdisteitä, mu- kaan lukien niiden aktiivisuus, biosynteesi ja toimintamekanismi. Tutkimuksessa hyödyn- nettiin Helsingin yliopiston syanobakteerikantakokoelmaa. Syanobakteerikantoja seulottiin erityisesti niiden antileukeemisten ja antifungaalisten ominaisuuksien osalta käyttäen solu- viljelytestejä. Massaspektrometrisiä menetelmiä käytettiin yhdisteiden tunnistamiseen. Työssä havaittiin useita aktiivisia syanobakteerikantoja, joilta löydettiin mahdollisia uusia yhdisteitä. Lisäksi löydettiin useita tunnettuja yhdisteitä ja niiden aiemmin tunnistamatto- mia variantteja uusilta syanobakteerikannoilta tai uusista elinympäristöistä. Seulontatutkimusten perusteella valittiin syanobakteerikannat kokogenomisekven- sointiin. Genomianalyysin avulla pystyttiin selvittämään biosynteesireitti tunnetuille syto- toksisille yhdisteille nimeltä swinholidi ja skytofysiini. Lisäksi swinholidin tuotto pystyttiin osoittamaan ensimmäisen kerran laboratoriossa kasvatetussa puhtaassa syanobakteerikan- nassa. Väitöskirjan viimeisessä osatyössä tutkittiin tunnetun yhdisteen hassallidiinin toimintamekanismia, missä hyödynnettiin solutestejä, kuvantamista ja lipidimallinnusta. Anabaena-syanobakteerista puhdistetun hassallidiinin havaittiin hajottavan kolesterolia si- sältävien eukaryoottisolujen solukalvon. Luonnonyhdisteen kehittäminen lääkkeeksi tai muuksi lopputuotteeksi on hyvin hi- dasta, mutta tarpeellista tulevaisuuden kannalta. Tämä väitöskirjatutkimus lisää ymmärrys- tämme useista syanobakteerien tuottamista bioaktiivisista yhdisteistä sekä toiminnallisella että molekulaarisella tasolla.

VII

1 Introduction

1.1 Natural products and their importance Natural products are compounds produced in nature by micro- or macroorganisms. They are present from the bottom of the ocean to your backyard. A huge variety of places, sources and specific organisms have been studied for novel natural products, but even more striking and varied are the discovered chemical structures and features of these molecules. They vary from small to large, linear to cyclic, and certain molecule type to hybrids of several types, and many of them possess biological activities. Natural products have activities against many targets, including multiple mammalian and cancer cell lines, bacteria, viruses, parasites and more. Consequently, natural products are an important source of pharmaceuticals (Newman & Cragg 2016). In addition, natural products have found their way into many agricultural and nutritional applications as well as into chemical probes in research and industry (Cantrell et al. 2012; Singh et al. 2017). The era of natural product discovery started to flourish by the finding of antibiotics and the success of penicillin in the 1940s (Davies & Davies 2010; Giddings & Newman 2013). The discovery of penicillin proved the value of natural products and raised interest in finding more. Today, most of the drugs on the market are natural products or derived from them (Newman & Cragg 2016). These include a wide variety of drugs, not just antibiotics. From the year 1981 to 2014, 174 new anticancer and 32 antifungal drugs with novel chemistry were approved to the market, and among these, most of the anticancer drugs were natural product derived compounds (17 being unmodified natural product structures) (Newman & Cragg 2016). In contrast, the majority of accepted antifungal agents were synthetic in origin (Newman & Cragg 2016). Finding cures to diseases has been a principal goal in natural product discovery, with human interest guiding the research. The strong interest to find new drugs does not show signs of diminishing but rather the opposite, as antibiotic resistance is increasing as well as the incidence of cancers with insufficient treatment options. In recent years, producing antibody conjugate drugs, where a natural product is attached to an antibody that targets a certain tissue or tumor cells, has gained considerable interest (Beck et al. 2017). Such combination techniques provide new prospects also for those natural products that, for instance, display unacceptable levels of toxicity. Over the years, scientists and the industry have shown interest in natural products not only for use as pharmaceuticals but also for use in research and biotechnological applications. Nevertheless, their role in nature is also worthy of attention. Natural products, also called secondary metabolites or specialized metabolites, are not usually considered essential for the growth or reproduction of the producing organism. However, they can play an important role in the survival and distribution of the organism. For instance, they may act as signaling molecules between organisms or with the environment, or represent defense or predation mechanisms for the organism. Information regarding produced compounds, the targets of attack or defense, and the functioning of compounds in their own environ-

1 ment, is often lacking. However, knowledge regarding ecological aspects may help to identify active parts of the compound, improve productivity or even find novel compounds. In addition to providing benefits to producer organisms, natural products may be useful to surrounding organisms. Many examples of such cooperation exist, such as the symbiosis within sponges or lichens. Natural products are assembled from small building blocks, often via ribosomal or nonribosomal biosynthesis pathways. The biosynthesis pathways are composed of individual enzymes, which together produce the chemical structure and properties of a natural product and further enable variation and production of multiple variants of the natural product (Firn & Jones 2000; Hertweck 2009; Marahiel 2016). The identification of biosynthesis pathways and sequencing whole genomes have greatly altered the focus of natural product discovery. Currently, genome-mining projects are undertaken alongside traditional bioactivity screening studies. Genetic tools have reaccelerated natural product discovery after experiencing a slump, including abandonment by several pharmaceutical companies. However, finding active hits is still a difficult task as only a fraction of compounds are active or useful (Firn & Jones 2000). Nevertheless, the omics era has proven that there is still much to discover. For example, Actinobacteria have been an important source of natural products for a long while but genomic studies have shown that they still harbor previously unidentified biosynthesis gene clusters (see e.g. Bentley et al. 2002). Other prolific bacterial sources include Proteobacteria, Firmicutes, and Cyanobacteria but also fungi belonging to Ascomy- cota contain vast amounts of natural product biosynthesis pathways (Wang et al. 2014, 2015). Thus, microbes have remained a key target for searching natural products. One microbial species may produce a number of different compounds and have many biosynthesis pathway genes in its genome. Furthermore, bioinformatic methods can reveal the evolutionary origins of natural product diversity in nature and horizontal gene transfer (HGT) events which have been shown in many cases (Jensen 2016; Ziemert et al. 2016). Altogether, natural products play a crucial part in our lives as well as in the lives of microbes. Vast amounts of different bioactivities have been described from these natural products but many compounds are yet to be discovered. In addition to scientific curiosity, the human utility perspective drives natural product discovery. Important aspects of natural product research include identifying chemical structures and biosynthesis genes, and evaluating and testing the activities and mechanisms of action of the compounds.

1.2 Cyanobacteria and their natural products Cyanobacteria are oxygenic photosynthetic prokaryotes comprising a single phylogenetic branch in the domain Bacteria (Komárek et al. 2014; Castenholz 2015). These photoauto- trophic bacteria evolved early in Earth's time scale, and are believed to have influenced the rise of oxygen levels in the atmosphere between 2.5 and 2.3 billion years ago (Bekker et al. 2004; Schopf 2012). Cyanobacteria might even have existed much earlier (Schirrmeister et al. 2015). During their long history, cyanobacteria have spread to a diverse range of environments, and they exist in various forms, morphologically varying from unicellular single

2 cells to branching filaments (Whitton & Potts 2012; Komárek et al. 2014; Castenholz 2015). A number of cyanobacteria live as planktonic organisms in oceans, seas and fresh waters (Whitton & Potts 2012). These water environments are inhabited also by benthic cyanobacteria, which are attached to surfaces such as rocks, plants and animals. Moreover, cyanobacteria are found from terrestrial, symbiotic and extreme habitats including hot springs, and hot and cold deserts (Whitton & Potts 2012; Cirés et al. 2017). Besides the oxygenic photosynthetic properties of cyanobacteria, many of them can fix nitrogen (Castenholz 2015). Cyanobacteria have specialized cells, heterocytes, for nitrogen fixation. Cyanobacte- ria may also produce other specialized cells such as akinetes (resting cells) or hormogonia (chain of migrating cells) (Castenholz 2015). Furthermore, cyanobacteria produce various natural products (Burja et al. 2001; Welker & von Döhren 2006; Singh et al. 2011). Cyanobacteria have gained widespread interest in recent years. Their photosynthetic machinery, various natural products and adaptation to various environments provide many avenues for applications. Cyanobacteria are sold as a health food supplement with great success, and they are used in feedstock and in agriculture as a soil additive (Abed et al. 2009; Mazard et al. 2016; Singh et al. 2017). One of the main areas of interest is currently biofuel production (Mazard et al. 2016; Singh et al. 2017). The current interest in cyanobacteria, however, started from toxins. Cyanobacterial blooms (example in Figure 1) are a considerable risk for humans and animals all around the globe, including the Baltic Sea and fresh water environments (Sivonen et al. 1989, 2007; Carmichael 2001; Codd et al. 2005a,b). Unfortunately, toxin production cannot be easily distinguished from blooms. Toxic cyanobacteria cause human and animal poisonings, shellfish poisoning, problems in fishery, and skin irritation (swimmer's itch) to recreational users (Codd et al. 2005a; Sivonen 2009). Cyanobacteria also cause aesthetic harm owing to water odour and taste problems (Watson 2003; Codd et al. 2005b). The human liver toxin microcystin, which is often associated with bloom forming cyanobacteria, is the most extensively studied cyanobacterial natural product (Carmichael et al. 1988; Sivonen 2009). Other cyanobacterial toxins include anatoxin, aplysiatoxin, cylindrospermopsin, nodularin, and saxitoxin (Codd et al. 2005b; Sivonen 2009).

Figure 1. Cyanobacterial bloom in Finnish coastal area of Baltic Sea (Laajasalo, Helsinki, July 2018). 3 Besides toxins, many other bioactive natural products have been found from cyanobacteria (Burja et al. 2001; Singh et al. 2011; Gerwick & Moore 2012), and already by the 1980s, many compounds had been described from cyanobacteria, including dolastatin and scytophycin (Pettit et al. 1981; Ishibashi et al. 1986). Cyanobacterial natural products are structurally variable, and include compounds such as peptides, polyketides, alkaloids, and ter- penes (Welker & von Döhren 2006; Jones et al. 2009; Dittmann et al. 2015; Yamada et al. 2015). Modern genomic techniques support the conclusion that cyanobacteria are rich in natural products, and enable an increasing number of compounds to be identified (Calteau et al. 2014; Wang et al. 2014, 2015; Micallef et al. 2015). Especially marine cyanobacteria have been a target of interest (Burja et al. 2001; Tan 2007; Nunnery et al. 2010; Blunt et al. 2017). One of the most extensively studied marine cyanobacterial species rich in natural products is Moorea (previously Lyngbya sp.), which produces compounds such as curacin, jamaicamide, and apratoxin (Kleigrewe et al. 2016; Leao et al. 2017). Fresh and brackish water species and terrestrial cyanobacteria produce bioactive molecules as well (Welker & von Döhren 2006; Chlipala et al. 2012). These include the trypsin inhibitors nostosin (Liu et al. 2014c), pseudospumigin (Jokela et al. 2017), and suomilide (Fujii et al. 1997); the cytotoxic compounds (i.e. compounds toxic to cells) anabaenolysin (Jokela et al. 2012) and scytophycin (Ishibashi et al. 1986); and the antifungal compound hassallidin (Neuhof et al. 2005).

1.3 Bioactivity The numerous natural products produced by cyanobacteria vary greatly in structure and therefore also in biological activity. Cyanobacterial natural compounds exhibit antibacterial, antiprotozoal, antiviral, antifungal, and cytotoxic activity (Burja et al. 2001; Tan 2007; Singh et al. 2011; Swain et al. 2017). In addition, some have protease or ion channel inhibitory activity. However, certain natural products have no described activity. These observations have led to a debate regarding whether natural products confer a fitness advantage to the host (Firn & Jones 2000; Jensen 2016). There are likely several evolutionary paths creating the numerous natural products. The discussion can be further complicated by limited knowledge regarding the activity of natural products. The identified activity is only based on the bioactivity assay performed, and the more the compound is tested, the more activities may be revealed. For instance, anticancer compounds show commonly also antifungal activity, thus being toxic or inhibitory to all eukaryotic cells. Many bioactive natural compounds from cyanobacteria have been tested in cell assays under specific conditions, and the ecological roles of the active compounds remain unknown. Surprisingly little is known about why cyanobacteria produce bioactive compounds. However, some advantage for the producer is expected (Méjean & Ploux 2013). One hypothesis is that cyanobacteria compete with other organisms and the bioactive natural product gives them a competitive advantage. For example, tolytoxin production was found to increase in the cyanobacterium Scytonema ocellatum in the presence of fungal cell wall

4 components, indicating that the production of this cytotoxic compound is a defense mechanism against co-occurring fungal species (Patterson & Bolis 1997). In addition, cyanobacteria have been found to inhibit the growth of other cyanobacteria, micro- and macro-algae and some plants, suggesting that certain compounds may have allelopathic activity (Leão et al. 2009). Cyanobacteria live in numerous environments in complex food webs with other organisms, including fungi and viruses, which could indicate that cyanobacteria need defense mechanisms but also molecules for communication between organisms. Cyanobacte- ria are often found in symbiosis, for example, with lichens and marine sponges. However, only photoprotective agents such as scytonemin have thus far been shown to have a clear role in symbiosis, protecting cyanobacteria and their symbionts from harmful UV light (Garcia-Pichel & Castenholz 1991; Nguyen et al. 2013a). Regardless of their ecological functions, the diverse and potential activities of cyanobacterial natural products have caused them to gain interest as drug candidates. The following chapters provide examples of cyanobacterial anticancer and antifungal natural products.

1.3.1 Anticancer activity New anticancer drugs are needed due to the increasing prevalence of cancer and the diversity of the cancers detected. Furthermore, tumor cells are developing resistance against drugs, similar to bacteria against antibiotics. In many cases, cancer treatment involves heavy chemotherapy, and there is a pressing need for new drugs. A number of cyanobacterial natural products show anticancer or cytotoxic activity (see examples in Figure 2 and Table 1). Some cyanobacterial anticancer compounds or analogues of them have entered clinical trials but only one compound has thus far made it to the market. In 2011, FDA approved Adce- tris® (Brentuximab vedotin, Seattle Genetics®) for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma (with certain regulations) (Deng et al. 2013). In 2012, the drug was also approved in Europe, and in 2018, the spectrum of treated Hodg- kin's lymphomas was expanded. The antibody-drug conjugate Adcetris® consists of an anti- CD30 monoclonal antibody and the cytotoxic monomethyl auristatin E, which is a synthetic analogue of dolastatin 10 (Figure 2) (Deng et al. 2013). Dolastatins were first described from the sea hare Dolabella auricularia (Pettit et al. 1981, 1987) but the actual producer was later found to be the cyanobacterium Symploca sp. (Luesch et al. 2001a). Dolastatins show activity against several cancer cell lines, the variants dolastatin 10 and 15 showing the highest potential (Pettit et al. 1987, 1989). Several variants and analogues of dolastatins have been tested and entered clinical trials but the monomethyls auristatin E and F seem to be the most potent drug candidates, and there are several ongoing studies on these variants as antibody-drug conjugates against different cancer types (Mayer 2018). Another anticancer peptide from cyanobacteria is apratoxin A (Figure 2) (Luesch et al. 2001b). It was isolated from Lyngbya majuscula and shows activity against several solid tumor cell lines (Luesch et al. 2001b). In addition, other apratoxin variants show cytotoxic activity (Luesch et al. 2002; Gutiérrez et al. 2008). Many Moorea sp. (previously named Lyngbya sp.) have been found to produce bioactive compounds (Engene et al. 2012; Leao 5 et al. 2017), such as curacin A (Figure 2) (Gerwick et al. 1994). Curacin A shows antimitotic activity but is also a potent brine shrimp toxin (Gerwick et al. 1994). However, the development of curacin A into a drug has been hindered by its insolubility. The macrolide compounds cryptophycins (Figure 2) were found from Nostoc sp. GSV 224 (Schwartz et al. 1990; Trimurtulu et al. 1994). Cryptophycin A shows antifungal activity and is active against several cancer cell lines at the picomolar range (Schwartz et al. 1990; Smith et al. 1994; Trimurtulu et al. 1994). Synthetic variants of cryptophycin have been produced and extensively studied, with cryptophycin 52 being a particularly promising drug candidate (Edelman et al. 2003). Another group of cyanobacterial macrolide compounds is the scytophycins (Figure 2), which include several variants such as tolytoxin. Scytophycins have been isolated from Scytonema spp., Cylindrospermum, and Nostoc sp. (Ishibashi et al. 1986; Carmeli et al. 1990; Jung et al. 1991; Tomsickova et al. 2014). Scytophycins show potent cytotoxic effects against human cancer cell lines as well as against fungi (Patterson & Carmeli 1992). Scytophycins are structurally similar to swinholides, which are associated with symbiotic sponge species although their actual producer is unknown. However, the swinholide variants ankaraholides have been described from a field sample containing the cyanobacterium Geitlerinema sp. (Andrianasolo et al. 2005). Swinholides show cytotoxic and antifungal activity similar to scytophycins (Carmeli & Kashman 1985; Kobayashi et al. 1990). Many of the anticancer compounds mentioned above were screened with the US Na- tional Cancer Institute (NCI) 60 human tumor cell line anticancer drug screen (Shoemaker 2006). This panel includes a few leukemia cell lines but no acute myeloid leukemia (AML) cell lines. AML treatment is also in urgent need of new drugs as the drugs currently used to treat acute myeloid leukemia were developed half a century ago (Rowe 2013). Not many screening studies have been carried out with cyanobacteria and AML cells. However, a potential for apoptosis (i.e. programmed cell death typically involving e.g. blebbing or nuclear fragmentation) inducing activity in AML cells has been detected in compounds produced by cyanobacterial strains from certain locations and habitats including the Baltic Sea and lichen associated cyanobacteria (Herfindal et al. 2005; Oftedal et al. 2010, 2011; Liu et al. 2014a).

6 Figure 2. Chemical structures of some anticancer and/or cytotoxic natural products produced by cyanobacteria.

7 Table 1. Examples of anticancer (or cytotoxic) natural products found from cyanobacteria. Most of the example compounds have several variants, often separated by different letters.

Compound Chemical structure Cyanobacterial taxa Reference Almiramide Linear lipopeptide Lyngbya majuscula Sanchez et al. 2010 Ankaraholide Glycosylated polyketide Geitlerinema sp. Andrianasolo et al. 2005 Apratoxin Cyclodepsipeptide (mixed NRPS- Lyngbya sp. Luesch et al. 2001b PKS) Aurilide Cyclodepsipeptide (mixed NRPS- Lyngbya majuscula Suenaga et al. 1996; Han PKS) et al. 2006 Belamide Linear tetrapeptide Symploca sp. Simmons et al. 2006 Bisebromoamide Linear heptapeptide Lyngbya sp. Teruya et al. 2009 Calothrixin Indole alkaloid Calothrix sp. Rickards et al. 1999 Carmaphysin Linear tripeptide Symploca sp. Pereira et al. 2012 Caylobolide Macrolactone polyketide Lyngbya majuscula MacMillan & Molinski 2002 Coibamide Cyclodepsipeptide Leptolyngbya sp. Medina et al. 2008 Cryptophycin Macrolactone (mixed NRPS-PKS) Nostoc sp. Schwartz et al. 1990 Curacin Linear peptide (mixed NRPS-PKS) Lyngbya majuscula Gerwick et al. 1994 Dolastatin Linear peptidic compounds Symploca sp. Pettit et al. 1981; Luesch et al. 2001a Dragonamide Linear lipotetrapeptide Lyngbya spp. Jiménez & Scheuer 2001; Gunasekera et al. 2008 Hectochlorin Cyclic mixed NRPS-PKS Lyngbya majuscula Marquez et al. 2002 Hoiamide Cyclic lipodepsipeptide (mixed L. majuscula, Phormidium Pereira et al. 2009 NRPS-PKS) gracile Jamaicamide Linear lipopeptide (mixed NRPS- Lyngbya majuscula Edwards et al. 2004 PKS) Lagunamide Cyclodepsipeptide Lyngbya majuscula Tripathi et al. 2010 Largazole Cyclic lipodepsipeptide Symploca sp. Taori et al. 2008 Lyngbyabellin Cyclic lipodepsipeptide Lyngbya majuscula Luesch et al. 2000 Malevamide Cyclic lipodepsipeptide, linear pep- Symploca laete-viridis Horgen et al. 2000 tides Malyngamide, Small linear amides Lyngbya majuscula Mynderse & Moore 1978; isomalyngamide Chang et al. 2011 Minutissamide Cyclic lipodecapeptide Anabaena minutissima Kang et al. 2011 Obyanamide Cyclodepsipeptide Lyngbya confervoides Williams et al. 2002 Palmyramide Cyclodepsipeptide Lyngbya majuscula Taniguchi et al. 2010 Pseudodysidenin Linear depsipeptide Lyngbya majuscula Jiménez & Scheuer 2001 Santacruzamate Small linear diamides Symploca sp. Pavlik et al. 2013 Scytophycin Macrolactone (mixed NRPS-PKS) Scytonema sp. Ishibashi et al. 1986 Somocystinamide Linear mixed NRPS-PKS compound Lyngbya majuscula Nogle & Gerwick 2002 Symplocamide Ahp-cyclodepsipeptide Symploca sp. Linington et al. 2008 Tubercidin Purine ribonucleoside Tolypothrix byssoidea Barchi et al. 1983 Veraguamide Cyclic lipodepsipeptide Oscillatoria margaritifera Mevers et al. 2011

8 1.3.2 Antifungal activity A variety of cyanobacteria produce natural products that exhibit antifungal activity (Abed et al. 2009; Chlipala et al. 2012; Swain et al. 2017). The chemical structures of these compounds are variable, including alkaloids, aromatic compounds, polyketides and different kinds of peptides. Examples of specifically antifungal natural compounds include fischerindole (Park et al. 1992), laxaphycin (Frankmölle et al. 1992), and welwitindolinone (Stratmann et al. 1994b). Furthermore, there are even more compounds that exhibit antifungal activity along with other activities. There are natural compounds showing, for instance, antifungal and cytotoxic activity against mammalian cells, such as scytophycins and swinholides (Carmeli & Kashman 1985; Kobayashi et al. 1990; Patterson & Carmeli 1992), or against other cyanobacteria, such as fischerellin (Gross et al. 1991). One interesting group of compounds is the glycolipopeptides hassallidins, which consist of a peptide ring of eight amino acids with one additional amino acid, fatty acid and one to three sugar moieties (Tables 2, 3 and 4). Hassallidin A, the first variant described, was isolated from the terrestrial epilithic cyanobacterium Tolypothrix sp. (basionym Hassallia) (Neuhof et al. 2005). Hassallidin A has only one sugar in its structure. It was shown to exhibit a minimum inhibitory concentration (MIC) of 4.8 μg ml-1 (3 μM) against Candida albicans and Aspergillus fumigatus (Neuhof et al. 2005). In addition, clear inhibition zones were seen in plate diffusion assays against other fungi, including Aspergillus niger, Candida glabrata, Fusarium sambucium, Penicillium sp., and Ustilago maydis (Neuhof et al. 2005). Hassallidin B (isolated from the same strain), in turn, has two carbohydrate units, which have little effect on the activity of the compound (Neuhof et al. 2006b). The MIC values against several strains of Candida sp. and Cryptococcus neoformans were shown to range from 8 to 16 μg ml-1 (5 to 10 μM) for hassallidin B and from 4 to 16 μg ml-1 (3 to 12 μM) for hassallidin A (Neuhof et al. 2006b). Hassallidin A and B were subsequently patented, and additional antifungal testing was carried out in addition to cytotoxicity assays (Neuhof et al. 2006a). -1 Hassallidin B showed a half-maximal inhibitory concentration (IC50) of 0.2 μg ml (0.1 μM) against the Jurkat ATCC-TIB-152 (human acute T cell leukemia) cell line, and both hassallidin A and B showed high activity against L 929 (murine aneuploid fibrosarcoma) cells (resazurin assay) (Neuhof et al. 2006a). The biosynthesis genes for hassallidin production were described from the Baltic Sea isolate Dolichospermum sp. UHCC 0090 (previously Anabaena sp. 90), which led to the discovery of many new producer genera and locations (Table 3) (Wang et al. 2012; Vestola et al. 2014). Furthermore, the main variants hassallidin C and D were described and shown to have similar antifungal activity (Vestola et al. 2014). For hassallidin D, an MIC value of -1 -1 2.8 μg ml (2 μM) and IC50 values of 0.55 to 1.86 μg ml (0.3 to 1 μM) were measured against different C. albicans strains (Vestola et al. 2014). The most recently described main variant is hassallidin E, which was isolated from the nonheterocystous cyanobacterium Planktothrix serta PCC 8927 (Pancrace et al. 2017b). By then, hassallidins and their variants balticidins had been found only from heterocystous species such as Anabaena/Dolichosper- mum, Aphanizomenon, Cylindrospermopsis, Nostoc and Tolypothrix spp. (Table 3) (Neuhof et al. 2005; Bui et al. 2014; Vestola et al. 2014). In addition, many other variants had been 9 detected but not described as main variant categories (Vestola et al. 2014). Interestingly, hassallidins are now being increasingly found from across the world due to expanding genomic data (Abreu et al. 2018). The terms hassallidin or balticidin are used for these glycolipopeptides when found from cyanobacteria but structurally identical or highly similar compounds are found with inconsistent naming from other bacteria (Table 4), including herbicolin (Aydin et al. 1985), jagaricin (Graupner et al. 2012), chromobactomycin (Kim et al. 2014), and Sch 20561 and 20562 (Afonso et al. 1999a,b). They exhibit antifungal activities highly similar to hassallidins. Moreover, similar to hassallidins, no antibacterial activity has been shown for these compounds (Neuhof et al. 2005), with the exception of herbicolin A, which displays activity against mycobacteria (Freundt & Winkelmann 1984). Cyanobacteria produce surprisingly many peptides with lipid moieties of varying lengths attached to them (Burja et al. 2001). Many of these lipopeptides have cytotoxic activities. For instance, the linear lipopeptide malyngamide exhibits cytotoxic activity (Appleton et al. 2002; Sabry et al. 2017; Jiang et al. 2018) and dragonamide shows antipar- asitic activity (Balunas et al. 2010). Furthermore, besides the hassallidins, cyanobacteria also produce other cyclic lipopeptides (examples in Table 5). Anabaenolysins (Jokela et al. 2012), calophycins (Moon et al. 1992), laxaphycins (Frankmölle et al. 1992), lobocyclamides (MacMillan et al. 2002), lyngbyacyclamides (Maru et al. 2010), muscotoxins (Tomek et al. 2015), and puwainaphycins (Hrouzek et al. 2012) are all examples of cyclic lipopeptides ex- tracted from cyanobacteria and exhibiting cytotoxic or antifungal activities. For example, anabaenolysins comprise a group of compounds found uniquely from benthic Anabaena strains in coastal areas (Gulf of Finland, Baltic Sea) (Jokela et al. 2012; Shishido et al. 2015).

10 2017b . 2005 . 2006b . 2014 . 2014 . 1999b . 1999a et al. . 2014** et al et al et al et al et al et al et al . 1985 et al . 1985 et al et al . 2014 et al . 2014 et al . 2014 et al . 2014 tola Reference + 5,64 Kim 82,69 Neuhof 10,73 Pancrace 1528,75 Neuhof Monosaccharides w that D-aa is in position 7. ln GalA Ara Man 1734,79 Bui ln-OH GalA Ara Man 1752.80 Bui Amino acids sallidin structure after reanalyzing HMBC data. der as both chemical and biosynthesis gene cluster evidence sho -Thr D-Tyr -Thr E-Dhb D-Gln D-Tyr Gly L-NMe-allo-Thr E-Dhb D-Gln Gly L-NMe-allo-Thr L-His L-His - - - - - -D-Glc 1357,69 Afonso 1195,64 Afonso -Thr D-Leu -Thr Gly D-Leu -Thr D-Gln Gly Gly D-Tyr L-NMe-allo-Thr D-Gln Gly Dhb L-NMe-allo-Thr L-Arg D-Gln Gly L-Arg L-allo-Thr - L-His - D-Glc - - - - 1300,74 - Aydin 1138,68 - Aydin 1181,62 Graupner et al. 2012 -Thr D-Tyr -Thr Dhb D-Tyr D-Gln Gly Dhb D-Gln Gly NMeThr NMeThr Lys L-Tyr GlcNAc GlcNAc Ara Ara diAcMan diAcMan 1829,90 Vestola 1864,87 Ves allo allo allo allo allo allo allo 1 2 3 4 5 6 7 8 9 10 M1 M2 M3 [M+H] Fatty acid 2,3-OH-C16 Thr Thr Thr Tyr(3-OH) Dhb L-Gln Gly NMeThr D-G 2,3-OH-C16 Thr Thr Thr Tyr(3-OH) Dhb L-Gln Gly NMeThr D-G D-3-OH-C14 D-3-OH-C14 E-Dhb L-Thr D- E-Dhb L-Thr D- (R)-3-OH-C14 Dhb L-Thr D- Sch 20561 Sch 20562 Herbicolin AHerbicolin BJagaricin (R)-3-OH-C14 Chromobactomycin DL-Dhb L-Thr (R)-3-OH-C14 D- 3-OH-C14 DL-Dhb L-Thr D- Dhb Thr Thr Tyr Dhb Gln Gly NMeThr His - - - 119 Name Balticidin D Hassallidin AHassallidin B 2,3-OH-C14 2,3-OH-C14 Thr Thr Thr Thr Thr Thr Tyr Tyr Dhb Gln Dhb Gly Gln Gly NMeThr NMeThr Gln Gln - - - Rha Man Man 13 Hassallidin CHassallidin D 2,3-OH-C16 2,3-OH-C16 L-Thr Thr L-Thr D- Thr D- Balticidin A*Balticidin BBalticidin C 2,3-OH-13-Cl-C16 Thr 2,3-OH-13-Cl-C16 Thr Thr Thr Thr Tyr(3-OH) Thr Dhb L-Gln Gly Tyr(3-OH) Dhb L-Gln NMeThr Gly NMeThr D-Gln-OH GalA D-Gln Ara GalA Man Ara 1786.77 Bui Man 1768,76 Bui Hassallidin E 2,3-OH-C16 Thr Thr Thr Tyr Dhb Gln Gly NMeThr Gln - - Hex 14 Table 2. Hassallidin family glycolipodepsinonapeptides. Ac, acetyl; Dhb, dehydrobutyric acid; NMe, N-methyl * The balticidin amino acids 7 and 10 are likely in opposite or ** The sequence of amino acids 5 to 10 was changed to match has 11 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al udy a l o t s e estola estola +Vestola analysis Reference Chemical + - Vestola +++ ++ Vestola + + Vestola Vestola + V + + Vestola + + Vestola + + Vestola + + Vestola + + Vestola + + Vestola + - Vestola + + Vestola NA + T his study NA + This st Genes The variants are listed to provide an overview of to provide an overview The variants are listed ers + + Vestola others + + Vestola others + + Vestola D, othersD++V + + Vestola idin B ticidin B lticidin B, hassallidin C + + Vestola assallidin C, others Hassallidin C, others Hassallidin C, others Variants (identical or relatives) Others Hassallidin C, B, others + + V Hassallidin C, D, others +* Hassallidin C, D, others Hassallidin C, D Hassallidin C, others Hassallidin D Hassallidin C, D, oth Hassallidin C, D, Hassallidin C, Hassallidin C, Others Hassallidin C, D, Hassallidin C, D Lake Frøylandsvatnet, Norway Lake Frøylandsvatnet, Norway Lake Vesijärvi, Finland Lake Tuusulanjärvi, Finland Lake Vesijärvi, Finland Origin Lake Tuusulanjärvi, Finland The Gulf of Finland, Finland The Gulf of Finland, Vuosaari, Finland Baltic Lake Vesijärvi, Finland Lake Knud, Denmark Kobben, Hanko, Baltic Sea, Finland Lake Tuusulanjärvi, Finland sp. UHCC 0404 (SYKE 971/6) Lake Kotojärvi, Finland UHCC 0090 (90) UHCC 0258 (258) UHCC 0229 (XPORK 5C)UHCC 0233 (XSPORK 7B) Porkkala Cape, Baltic Sea coast, Finland Porkkala Cape, Baltic Sea coast, Finland Bal Ba UHCC 0406 (SYKE 748A)UHCC 0420 (SYKE 763A) Lake Tuusulanjärvi, Finland Lake Tuusulanjärvi, Finland UHCC 0780 (HAN 7/1) UHCC 0299 (299A) UHCC 0298 (299B) UHCC 0488 (0TU43S8) UHCC 0476 (0TU33S16)UHCC 0481 (1TU 33S8)UHCC 0487 (1TU 35S12)UHCC 0486 (1TU 44S9) Lake Tuusulanjärvi, Finland 1TU 44S16 Lake Tuusulanjärvi, Finland Lake Tuusulanjärvi, Finland UHCC 0688 (BIR JV1) UHCC 0172 (BECID19) Lake Tuusulanjärvi, Finland UHCC 0261 (XSPORK 14D)UHCC 0248 (XSPORK 36B) Porkkala Cape, Baltic Sea coast, Finland Porkkala Cape, Baltic Sea coast, Finland Others H PH 256 NIVA-CYA 269/6 NIVA-CYA 269/2 Strain and name Anabaena sp. / Dolichospermum variant diversity (identical or related to main variants). In addition, the identification of production through genes or chemical analysis is shown (+, positive or chemical analysis is shown through genes identification of production main variants). In addition, the or related to variant diversity (identical NA, data not available). -, no variants found; for genes or chemical analysis; Table 3. List of all known cyanobacterial strains producing hassallidins A to E, balticidins or other variants. producing hassallidins Table 3. List of all known cyanobacterial strains 12 . 2006b . 2014 . 2014 . 2014 . 2005 . 2014 . 2014 . 2014 . 2014 . 2014 . 2018 . 2018 . 2018 et al . 2014 et al et al et al et al et al et al et al et al et al. et al et al et al et al ace et al ui Pancr 2017b analysis Reference Chemical +NAAbreu + - Vestola + + V estola +NAAbreu + + Vestola + + Vestola + + Vestola + NA Abreu + + Vestola + - Vestola + + Vestola NA + Neuhof NA + Neuhof NA + B NA + This study NA + study This Genes + + din B Balticidin B, Others Hassallidin A Hassallidin B others Others Variants (identical or relatives) Balticidin A, B, C, D Baltici Hassallidin E Others Balticidin B Hassallidin C, balticidin B, Others Aquarium, Stocholm, Sweden Lake Haukkajärvi, Finland Soil, Borneo Origin Sewage plant, Berre-le-Clos, France Watkins Glen State Park, NY, USA Theobaldo Dick Lake, Lajeado, RS, Brazil Lichen associated, Finland Baltic Sea, Rügen Island, Germany Epilithic, Bellano, Italy Epilithic, Bellano, Italy Freshwater lake, Singapore Morro Branco, CE, Brazil Lake Balaton, Hungary Pond, Townsville, NQ, Australia Freshwater, Solomon Dam, Q, Australia Others Dobre Pole, Czech Republic genes has resulted in inability to produce hassallidin. sp. has Bio33 PCC 7504 B02-07 B02-07 CENA 303 113.5 PCC 7101 Heaney/Camb 1986 140 1/1 Freshwater, Lough Neagh, Ireland ATC-9502 PCC 9009 CR12 PCC 8927 CS-508 CENA 219 CS-505 UHCC 0159 (159) 6 sf Calc sp. Aphanizomenon gracile Aphanizomenon raciborscii Cylinrdrospermopsis Tolypothrix Strain and name Planktothrix serta Nostoc *Mutation in 13 . 2012 . 1999b . 1999a . 1985 . 1986 et al . 2014 et al et al et al et al et al Graupner Afonso Afonso Kim Aydin Aydin alysis Reference + + + + + + NA NA NA NA NA sp. mushroom pathogen NA Associated environment Genes Chemical an Plant pathogens Plant pathogens Rhizobacteria sp. C61 A111 A112 sp. W-10 NRRL B-11053 Human pathogens sp. W-10 NRRL B-11053 Human pathogens Aeromonas Aeromonas Erwinia herbicola Erwinia herbicola Janthinobacterium agaricidamnosum Agaricus Chromobacterium Sch 20562 Sch 20561 Herbicolin A Herbicolin B Jagaricin Compound Bacterial strain Chromobactomycin Table 4. Hassallidin variants from other bacteria. Table 4. Hassallidin variants from other 14 1992 2014b et al. rated by different letters. et al. 1992 2002 1994a 2009 1998 2000; Liu 1992 2013 2005 2000 2009 2000 et al. 1992 et al. . 2012; Oftedal et al. 2012 et al. 2010 2004 2008 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. 2015 et al. 1989; Gregson et al et al. et al. et al. et al. et al. 2011 Reference Neuhof Moon Jokela Stratmann Pereira Gerwick Plaza & Bewley 2006 Taori MacMillan Frankmölle Luesch Horgen Maru Kang Moore Zainuddin Mevers Pergament & Carmeli 1994 Tomek Kajiyama Golakoti Berry sp. sp. sp. sp. sp. sp. sp. sp. Most of the example compounds have several variants, often sepa Cyanobacteria Calothrix fusca Anabaena Hapalosiphon welwitschii Lyngbya majuscula, Phormidium gracile Tolypothrix Hormothamnion enteromorphoides Oscillatoria sp. Symploca Lyngbya confervoides Anabaena laxa Lyngbya majuscula Lyngbya Lyngbya Symploca laete-viridis Anabaena minutissima Moorea producens Desmonostoc muscorum Nostoc commune Nostoc Lyngbya Anabaena Schizotrix sp. Activity Antifungal Cytotoxic Cytotoxic Cytotoxic Cytotoxic, antimicrobial Anticancer Cytotoxic Cytotoxic Cytotoxic Antifungal Unknown Antitoxin Antibacterial Anticancer Antimicrobial Cytotoxic Antifungal Cytotoxic Cytotoxic, antimicrobial Calophycin Anabaenolysin Compound Hapalosin Hoiamide Hassallidin Hormothamnin Largamide H Largazole Laxaphycin Lyngbyabellin Lobocyclamide LyngbyacyclamideLyngbyazothrin Malevamide Anticancer Minutissamide Precarriebowmide Cytotoxic Muscotoxin NostofungicidinePahayokolide Antifungal Puwainaphycin Schizotrin A Nostopeptolide Table 5. Examples of cyanobacterial cyclic lipopeptides. 15 1.4 Biosynthesis A biosynthetic gene cluster is a group of genes that encode the production of a natural product and are often genomically located close to each other (hence the word cluster). However, a biosynthetic gene cluster does not directly encode a natural product. Instead, the product is post-translationally modified or the genes encode enzymes that build the actual natural products. Cyanobacterial natural products are produced through ribosomal or nonribosomal biosynthesis pathways (Kehr et al. 2011). Nonribosomal peptides and polyketides are biosynthesized by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS) or hybrid pathways (NRPS-PKS). These pathways constitute the majority of identified cyanobacterial natural product biosynthetic pathways (Dittmann et al. 2015; Micallef et al. 2015). They are composed of multi-domain enzyme complexes that build the natural products from small precursor molecules into functional compounds by chain elongation (Fischbach & Walsh 2006). These pathways consist of modules, where each module produces one component to the growing molecule and moves it to the next module. The modules consist of smaller units called domains that are the enzymes modifying and moving the compound from one enzyme to another. Ribosomally synthetized peptides (RiPPs) are cleaved as precursor peptides, and the core peptide is modified post-translationally (Arnison et al. 2013). For instance, cyanobactins is a large family of cyanobacterial RiPPs (Sivonen et al. 2010). The biosynthetic gene clusters are often large and contain repetitive parts, and are therefore more prone to changes than the primary metabolites of the genome, which might explain their tremendous diversity (Medema et al. 2014; Ziemert et al. 2016). They are enriched in insertions, deletions and duplications, and horizontal gene transfer plays an important role in their evolution (Medema et al. 2014; Ziemert et al. 2016). Identification of natural product biosynthesis gene clusters is conceptually akin to genome mining, as the classical definition of genome mining is the search for specific biosynthetic genes for the production of a certain compound. The biosynthesis clusters found from cyanobacteria, which have genome sizes ranging from 1.4 to 12 Mb (e.g. Dolichospermum sp. UHCC 0090 has a 5.3 Mb genome (Wang et al. 2012)), vary in size from 2 to 58 kb (Méjean & Ploux 2013; Dittmann et al. 2015). The number of biosynthesis gene clusters correlates with genome size, such that cyanobacteria with smaller genomes may have less biosynthesis clusters (Shih et al. 2013). For instance, Cylindrospermum raciborskii genomes, which are the smallest known (approx. 3 Mb) among multicellular cyanobacteria, were found to have only a few biosynthesis gene clusters (Abreu et al. 2018). Rapidly increasing sequencing capacity and decreasing costs have enabled a huge increase in the availability of whole genomes in public databases and a preference for whole genome sequencing over targeting only biosynthesis genes. This has also lead to the development of multiple tools for the annotation of genomes or genes. The analysis of genetic data requires computing power and in silico tools. For example, the Minimal Information about a Biosynthetic Gene cluster (MIBiG) specification was developed to meet the needs of increasing biosynthetic gene cluster data (Medema et al. 2015). There are several repos- itories for the preservation of and access to genetic data, the most well-known being the

16 National Center for Biotechnology Information (NCBI) database of the U.S. National Library of Medicine. They also provide tools such as the Basic Local Alignment Search Tool (BLAST). One of the most well-known tools in natural product research today is antiSMASH that can be used to identify, annotate and analyze biosynthesis gene clusters from genomes (Medema et al. 2011; Blin et al. 2017). The development of genomic methods and tools has provided a major boost to natural product discovery. Genome mining is used to find, for instance, PKS, NRPS, RiPPs and terpenoid biosynthesis clusters using their conserved structures, but specific enzymes can also be used as search criteria. Nevertheless, results from genome mining are often limited to known biosynthesis gene cluster types, and it is still difficult to identify the genes responsible for the production of new structural classes. Het- erologous expression or knockout mutants are used to confirm that the predicted genes are responsible for producing the natural product. Genome mining and biosynthesis gene cluster identification often still depend on cultured strains, as metagenomes rarely produce the full biosynthesis gene cluster. Improvements in metagenomic analyses and methods such as sequence tagging and single-cell genomics are attempting to overcome this problem.

1.4.1 Polyketide synthesis Polyketides include compounds such as macrolides, polyphenols and polyenes. For example, macrolides consist of a macrocyclic lactone ring with additional side chains. Cyanobac- terial macrolides include compounds such as cryptophycin (Trimurtulu et al. 1994), lyngbyabellin (Luesch et al. 2000), and scytophycin (Ishibashi et al. 1986). Polyketide biosynthesis by a modular polyketide synthase resembles fatty acid synthesis (Figure 3). The common precursors for PKS include acetyl-CoA, malonyl-CoA, and propionyl-CoA (Hertweck 2009). The minimal PKS module consists of an acyltransferase (AT), an acyl carrier protein (ACP), and ketosynthase (KS) domains (Hertweck 2009; Kehr et al. 2011). The acyltransferase activates the acyl carrier protein and selects the precursor molecule. The acyl carrier protein moves the precursor into contact with ketosynthase, which is needed to produce the actual chain lengthening Claisen-condensation reaction. The chain-lengthening reaction is possibly followed by the action of modifying domains such as ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER). PKSs are commonly divided into three (I–III) subgroups (Hertweck 2009; Helfrich et al. 2014). Cyanobacterial PKSs belong into modular type I, which includes cis- and trans-AT PKSs. The cis-AT PKSs follow common co-linearity rules of biosynthesis pathways and they have iterative acyltransferases (AT) in each module. In contrast, the trans-AT PKSs have non-iterative acyltransferases, such that they use, for instance, only one AT multiple times (Hertweck 2009; Piel 2010). Other functions may also be provided itera- tively in trans-AT gene clusters, and they may have unusual domain orders, unique domains, non-elongating modules, or split modules (Piel 2010; Helfrich & Piel 2016). Interestingly, it has been proposed that cis- and trans-PKSs evolved independently from each other (Nguyen et al. 2008). Type II and III PKSs work in a completely iterative manner, and the products they typically synthetize are aromatic compounds (Hertweck 2009).

17 Figure 3. Modular polyketide synthase builds polyketides by a chain-elongation system. The loading module inserts the precursor (here, propionyl-CoA) and the following modules elongate the molecule. Additional enzymes, such as ketoreductase here, modify the growing molecule. Thioesterase often terminates chain lengthening and may cyclize the compound. AT, acyltransferase; KR, ketoreductase; KS, ketosynthase; TE, thioesterase; •, acyl carrier protein.

Despite the large amount of polyketides found from cyanobacteria, only a few biosynthesis gene clusters and their products have been identified (Wang et al. 2014; Dittmann et al. 2015). The cyanobacterial neurotoxins anatoxin-a and homoanatoxin-a are produced by type I cis-AT PKSs (Méjean et al. 2009, 2014; Rantala-Ylinen et al. 2011). The first trans-AT PKS gene cluster from cyanobacteria was identified from the symbiotic Nostoc sp. `Peltigera membranacea cymbiont` producing nosperin (Kampa et al. 2013). Furthermore, the tolytoxin gene cluster from Scytonema sp. PCC 10023 and luminaolide gene cluster from Planktothrix paucivesiculata PCC 9631 are produced by trans-AT PKSs (Ueoka et al. 2015). Recently, the biosynthesis gene clusters for phormidolide from Leptolyngbya sp. and nosperin-related cusperin from Cuspidothrix issatsechenkoi were shown to be produced by trans-AT PKSs (Bertin et al. 2016; Kust et al. 2018).

1.4.2 Nonribosomal peptide synthesis and hybrid synthesis Nonribosomal peptides and peptide-like compounds are biosynthesized through nonribosomal peptide synthetases (NRPS) or hybrid pathways of NRPS and PKS. Each NRPS module adds a single amino acid to the growing amino acid chain (Fischbach & Walsh 2006; Kehr et al. 2011; Marahiel 2016). The NRPS module needs an adenylation (A) domain for the activation and identification of the amino acid, a peptidyl carrier protein (PCP) to move the amino acid, and a condensation (C) domain to make the peptidic bond. Other tailoring domains may occur such as epimerization (E), methyltransferase (MT), and monooxygenase (OX) domains (Marahiel & Essen 2009; Marahiel 2016). In addition, a thioesterase (TE) domain often releases the final compound and terminates chain-elongation. Examples of cyanobacterial compounds produced through NRPS include aeruginosin (Ishida et al. 2009),

18 anabaenopeptin (Rouhiainen et al. 2010), and anabaenopeptilide (Rouhiainen et al. 2000). In addition, the antifungal compound hassallidin is produced through a NRPS (Wang et al. 2012; Vestola et al. 2014). Particularly many among cyanobacterial compounds are produced through NRPS-PKS hybrids, which consist of both PKS and NRPS domains or modules (Kehr et al. 2011). The hybrid biosynthetic gene clusters are important as many toxins such as microcystin and cylindrospermopsin are produced through them (Tillett et al. 2000; Mihali et al. 2008). In addition, many cytotoxic or anticancer natural products from cyanobacteria are produced by NRPS-PKS hybrids. These include the biosynthesis gene clusters of curacin A (Chang et al. 2004) and jamaicamide (Edwards et al. 2004), which are almost completely composed of PKS modules. In contrast, the anabaenolysin hybrid biosynthesis gene cluster has only one PKS module while the rest of the cluster consists of NRPS modules (Shishido et al. 2015). Other hybrid cyanobacterial biosynthesis routes include compounds such as apratoxin (Grindberg et al. 2011), barbamide (Chang et al. 2002), cryptophycin (Magarvey et al. 2006), hectochlorin (Ramaswamy et al. 2007), nodularin (Moffitt & Neilan 2004), nostocyclopeptide (Becker et al. 2004), nostophycin (Fewer et al. 2011) and puwainaphycin (Mareš et al. 2014).

1.5 Mechanism of action The mechanism of action (MOA) describes the interaction between a compound and its target. The study of a novel natural product often stops after describing its chemical structure and activity because of limitations regarding production (e.g. field samples) and purification of the novel compound. Thus, it is no surprise that despite the vast number of bioactive compounds found from cyanobacteria, only a few of them have studies on MOA. Stud- ies on MOA are important for understanding the ecological function of a compound and evaluating a compound for human derived applications (pharmaceutical, agricultural, industrial, etc.). Several different mechanisms of action have been described for cyanobacterial natural compounds (examples in Table 6), varying from non-targeted toxicity to small defined targets of a compound (Salvador-Reyes & Luesch 2015). Interruption of cytoskeletal structures, modulation of signaling pathways or enzymes, and interaction with DNA are examples of MOAs that have been associated with cyanobacterial compounds with anticancer activities (Costa et al. 2012; Tan 2013; Salvador-Reyes & Luesch 2015). Interruption of cytoskeletal structure in most cases signifies that the natural product targets actin or tubulin and, for example, depolymerizes the microfilaments. Several microfilament-targeting compounds have been found from cyanobacteria (Salvador-Reyes & Luesch 2015). These compounds include the cytotoxic compounds scytophycins and cryptophycins (Patterson et al. 1993; Smith et al. 1994). Scytophycins decrease the concentration of F-actin (Patterson et al. 1993) similar to another group of macrolide compounds, the swinholides (Bubb et al. 1995). However, there are several types of actin effects which may differ even between very closely related natural product variants (e.g. swinholide and

19 misakinolide, Terry et al. 1997). Inhibition of an enzyme or enzyme pathway is also a common MOA, and some compounds have been found to modulate ion channels (Salvador- Reyes & Luesch 2015). For example, apratoxin A inhibits protein translocation into the en- doplastic reticulum by targeting the Sec61 protein translocation channel (Paatero et al. 2016; Huang et al. 2016), whereas somocystinamide A activates the caspase-8-dependent cell death pathway in certain cancer cell lines (Wrasidlo et al. 2008). The hepatotoxin microcystin inhibits the PP1 and PP2A protein phosphatases (Honkanen et al. 1990). In addition, some cyanobacterial natural products are protease inhibitors, acting either on several different proteases or on a certain type. For example, nostosin is a trypsin inhibitor (Liu et al. 2014c), whereas anabaenopeptins and aeruginosins are large groups with varied inhibitor compounds (Itou et al. 1999; Kodani et al. 1999; Ersmark et al. 2008). The MOA may also be less specific as a compound might induce changes in the cell wall or cell membranes. Examples of cyanobacterial natural products with a wider target include the lipopeptides anabaenolysin and muscotoxin, which show detergent-type activities by disrupting the cell membrane (Oftedal et al. 2012; Tomek et al. 2015). These detergent-like cyanobacterial compounds act similar to other natural compounds found from plants and microbes, such as digitonin and surfactin, that destabilize membranes (Nishikawa et al. 1984; Carrillo et al. 2003). Anabaenolysin was found to affect outer cell membranes in a cholesterol-dependent manner (Oftedal et al. 2012), as opposed to muscotoxin, which was also found to affect membranes but without cholesterol (Tomek et al. 2015). The MOA of the antifungal compound hassallidin has remained unknown. However, hassallidin A has been compared to another cyclic lipopeptide, caspofungin (Neuhof et al. 2006c). The antifungal drug caspofungin is an example of echinochandin antifungals that target the fungal cell wall inhibiting -1,3-glucan synthase (McCormack et al. 2005). Hassallidin A was shown to lack this mechanism of action and was hypothesized to affect the cell membrane (Neuhof et al. 2006c).

20 Table 6. Examples of mechanisms of action of cyanobacterial natural products. The classification was adapted from Salvador-Reyes & Luesch (2015).

Mechanism of action and subclass Example compounds Reference Enzyme or pathway inhibition Acetylcholine receptor agonist Anatoxin A Carmichael et al. 1979 Glycosome inhibition Almiramides Sanchez et al. 2013 Histone deacetylase inhibition Largazole Ying et al. 2008 Santacruzamate A Pavlik et al. 2013 Kinase inhibition (signaling pathway) Bisebromoamide Teruya et al. 2009 Scytonemin Stevenson et al. 2002 Metal chelation Grassypeptolide Kwan et al. 2010 Oxidative stress inducer Calothrixin A Chen et al. 2003 Prohibitin inhibition Aurilide Sato et al. 2011 Protease inhibition Aeruginosinsc Ersmark et al. 2008 Anabaenopeptins Itou et al. 1999; Kodani et al. 1999 (e.g. G, H, T)b Cyanopeptolinsc Weckesser et al. 1996 Grassystatinsa Kwan et al. 2009 Largamidec Plaza & Bewley 2006 Pseudospumiginc Jokela et al. 2017 Suomilidec Fujii et al. 1997 Symplocamidec Linington et al. 2008 Symplocina Molinski et al. 2012 Tasiamide Ba Liu et al. 2012 Proteasome inhibition Carmaphycin Pereira et al. 2012 Protein phosphatase inhibition Microcystin Honkanen et al. 1990 Quorum sensing inhibition Malyngolide Dobretsov et al. 2010 Secretory pathway inhibition Apratoxin A Paatero et al. 2016; Huang et al. 2016 Somocystinamide Wrasidlo et al. 2008 Ion channel modulation Ion channel activation Antillatoxin Li et al. 2001 Hoiamide Pereira et al. 2009 Jamaicamide Edwards et al. 2004 Kalkitoxin Wu et al. 2000 Ion channel blocking Saxitoxin Catterall et al. 1979 Membrane disruption Cell surface disruption Anabaenolysin Oftedal et al. 2012 Muscotoxin Tomek et al. 2015 Microfilament targeting agents Actin-targeting Dolastatin 11 Bai et al. 2001 Hectochlorin Marquez et al. 2002 Lyngbyabellin Luesch et al. 2000 Puwainaphycin Hrouzek et al. 2012 Scytophycin Patterson et al. 1993; Smith et al. 1993 Swinholide Bubb et al. 1995 Tubulin-targeting Cryptophycin Smith et al. 1994 Curacin A Gerwick et al. 1994 Dolastatin 10 Bai et al. 1990 a Aspartic protease inhibition; b Exopeptidase (carboxypeptidase) inhibition; c Serine protease inhibition

21 2 Study aims

Especially prokaryotes and plants produce specialized metabolites, or so-called natural products. These compounds serve intriguing and complex purposes in the ecological food- webs of the organisms, displaying a diverse array of biological activities. Because of these features, many of these compounds show promise for use in human applications, including drugs. Most of the drugs in market today are natural products or derived from natural products. The need for new drugs is increasing because of drug resistance problems, and some diseases, especially cancers, still lack adequate treatments. In this study, I aimed to search for new antifungal and antileukemic compounds from cyanobacteria. Cyanobacteria have been found to be a prolific source of new natural products. From both the ecological and pharmaceutical points of view it is important to demonstrate which organisms produce these cytotoxic compounds, how they are produced, and how they function. In study I, I focused on identifying cyanobacterial strains with specific antileukemic activities. In study II, the antifungal activity was investigated. This investigation served as the basis for further identification and studies on active compounds. In study III, I aimed to describe the biosynthesis pathways of cytotoxic compounds identified in the first studies, namely swinholides and scytophycins. In study IV, the aim was to reveal the mechanism of action of the cytotoxic compound hassallidin. Examining several bioactive natural products, this study covered the first steps in natural product discovery from finding novel natural products to describing their production pathways and mechanisms of action.

The specific aims of this doctoral thesis were: . To screen cyanobacteria for antileukemic or cytotoxic activity and determine the role of their habitat or sampling place in the detected activity (I) . To screen cyanobacteria for the production of antifungal compounds (II) . To describe the swinholide biosynthesis gene clusters from Nostoc sp. and the scytophycin biosynthesis gene cluster from Anabaena sp. (III) . To determine the mechanism of action of the antifungal and cytotoxic compound hassallidin (IV)

22 3 Summary of materials and methods

3.1 Strains and cell lines used in this thesis All cyanobacterial strains were obtained from the University of Helsinki Cyanobacteria Cul- ture Collection (UHCC). The strains used in this thesis are listed in Supplemental Table 1 with their new names, as the naming system of the culture collection was unified during the study and strains were renamed using UHCC coding. The discovered swinholide and scytophycin biosynthesis gene clusters were submitted to NCBI GenBank with the accession numbers KY767987 and KY767986, respectively. The mammalian cell cultures used in this study included the human acute myeloid leukemia cell line MOLM-13 (Matsuo et al. 1997), the rat AML cell line IPC-81 (Lacaze et al. 1983), and the rat normal kidney cell line (NRK, ATCC CRL-6509). In addition, primary hepatocytes were isolated by in vitro collagenase perfusion from male Wistar rats or female C57BL/6JBomTac mice (Seglen 1976; Mellgren et al. 1995). The production of primary hepatocytes was performed in the laboratory of Stein Ove Døskeland (Department of Bio- medicine, University of Bergen, Norway). Otherwise, all mammalian cell lines were main- tained in the laboratory of Lars Herfindal (Department of Clinical Science, University of Ber- gen, Norway) following maintenance descriptions given in the publications. The cultured cells were tested every second month for mycoplasma infection with MycoAlert™ (Lonza Rockland, Inc., USA). The yeast Candida albicans in study IV was a patient-derived frozen culture main- tained at the Department of Clinical Science, University of Bergen (kindly provided by Audun Helge Nerland). The yeast C. albicans and fungus Aspergillus flavus used in study II origi- nated from the HAMBI Culture Collection (University of Helsinki).

3.2 Summary of methods The methods used in this doctoral thesis are summarized in Table 7. Detailed information regarding the methods is provided in the publications.

23 Table 7. Summary of methods.

Method Article Cell cultivation Cyanobacteria I, II, III, IV Mammalian cell lines I, IV Fungi II, IV Molecular methods PCR, cloning and sequencing of 16S rRNA I, II DNA extraction I, II, III Genome sequencing III Chemical methods Crude extract preparation I, II, III Purification of compound II, III, IV LC-MS I, II, III, IV NMR II, III, IV Bioactivity assays and mechanism of action Cytotoxicity assays I, IV Disc diffusion assays II Light and fluorescent microscopy I, IV Transmission electron microscopy IV Flow cytometric viability assays IV Liposome preparation and fluorescent assay IV Bioinformatics Phylogenetic and evolutionary analysis I, II, III Gene cluster identification III Molecular modeling IV

24 4 Summary of results and discussion

4.1 Bioactive cyanobacterial compounds Cyanobacterial natural products exhibit a wide range of activities, and they include many cytotoxic compounds (Singh et al. 2011; Gerwick & Moore 2012). Cytotoxicity can be measured in various ways, here represented by cytotoxicity assays with mammalian and yeast cells (I, II, IV). In study I, cyanobacterial crude extracts were screened for their activity against the human AML cell line MOLM-13, the rat AML cell line IPC-81, and normal mouse or rat hepatocytes to investigate anti-AML or nonspecific activity of cyanobacteria. Activity- guided search for novel compounds was also used in study II, where cyanobacteria were screened for antifungal activity. Both of these studies showed that any cyanobacterial strain, despite its habitat or genus, may show cytotoxic activity. The following structural identification and elucidation steps are often difficult but when accomplished, knowledge regarding these compounds may be considerably increased as was shown in the further studies (III, IV).

4.1.1 Antileukemic activity (I) Cyanobacterial compounds show diverse cytotoxic activities against many malignant cell lines. However, leukemia and particularly acute myeloid leukemia cells have not been tested extensively on cyanobacterial compounds or extracts. Here, among a set of 43 cyanobacterial strains, approximately 30% of the extracts showed selective activity towards acute myeloid leukemia cells (data set C). This is in line with previous results obtained from other sets of cyanobacteria (A and B) from UHCC (Herfindal et al. 2005; Oftedal et al. 2010; Liu et al. 2014a). The activity types in data set C varied considerably, and some cyanobacterial extracts even caused specific apoptosis or lysis of a certain AML cell line. Most of the activities found were present in organic extracts and only a few strains possessed activity in both aqueous and organic phase. Specific anti-AML activities were found from the cyanobacterial species Anabaena, Calothrix, Leptolyngbya, Nostoc, Planktothrix,andTrichormus. Known compounds were identified with mass spectrometric analysis from these cyanobacteria. These include compounds such as aeruginosin, anabaenopeptin, cyanobactin, depsipeptide, hassallidin, microcystin, and microviridin. Study I demonstrated that both the MOLM-13 and IPC-81 cell lines may be used to detect anti-AML activity. However, the MOLM-13 cells were more susceptible against cyanobacterial bioactive compounds (Table 8). Altogether, each data set (A–C, Table 8) demonstrated that cyanobacteria possess cytotoxic activities detectable with mammalian cell lines. If IPC-81 results from different data sets are compared, the amounts of active strains are very similar (Table 8). However, the earliest study (A) showed highest incidences of activity, which was hypothesized to be caused by the presence of adenosine in these extracts. Adenosine has been shown to induce apoptosis in some AML cell lines (Tanaka et al. 1994). A more comprehensive comparison of the data sets did not reveal any genus or sampling

25 Table 8. The number of cyanobacterial strains used in each data set for anti-leukemic testing and the number of strains showing over 70% cell death. In addition, the number of strains showing specific anti-AML activity is indicated.

Strains Strains Strains Strains showing Number showing showing showing specific anti- of cyano- over 70% specific anti- over 70% cell AML activity bacterial cell death AML activity death (MOLM-13 or Data sets strains (IPC-81) (IPC-81) (MOLM-13) IPC-81) Herfindal et al. 2005 A 43 14 (33%) 5 (12%) NA NA & Oftedal et al. 2010 B Liu et al. 2014 40 4 (10%) 2 (5%) NA NA

C Humisto et al. 2016 43 7* (16%) 2 (5%) 19** (44%) 13 (30%)

NA = Not available *Two strains lack specificity information (hepatocyte test) **Four strains lack specificity information (hepatocyte test) place to be superior with respect to anticancer activity profile. However, isolates from benthic habitats frequently displayed desired activities. The drawback of testing extracts is that the activity can result from the combined effect of several compounds, or change or disappear when tested with a purer compound. In addition, the identification of known compounds from a crude extract is time-consuming and difficult. There is also growing evidence that cyanobacteria simultaneously produce toxins and antitoxins, such as microcystin and small peptides (Jokela et al. 2010; Herfindal et al. 2011). All data sets (A–C) included several extracts which contained unknown compounds producing the activity. Purification of active compounds from extracts and further studies were initiated during the thesis but have not yet yielded candidate compounds for the treatment of AML. However, the previous data sets have provided other interesting natural products. One example is anabaenolysin whose structure and activity were determined after the anti-AML study (Oftedal et al. 2010; Jokela et al. 2012).

4.1.2 Antifungal or cytotoxic activity (II, IV) In study II, a large set of cyanobacterial strains (methanol extracts) was tested against the fungi Candida albicans and/or Aspergillus flavus using disk diffusion assay. Antifungal activity was detected only from 5.7% (11) of the 194 cyanobacterial extracts. Four of these strains produced hassallidins and another four scytophycins. Notably, the cyanobacterial strains produced a number of variants of hassallidins and scytophycins. Such variation occurs commonly in cyanobacterial natural products; for instance, the protease inhibitor compounds aeruginosins encompass a vast amount of variants (Ersmark et al. 2008; Liu et al. 2014c). From all of the eleven active hits detected in study II, three remained unidentified. Interestingly, one of the strains showing antifungal activity was earlier described to have antileukemic activity (Liu et al. 2014a). Here this cyanobacterium, Anabaena sp. UHCC

26 0451 (previously HAN 21/1), showed activity against both tested fungi. After further purification and analysis with spectrometric methods, the active compound was identified to be scytophycin, or specifically, many scytophycin variants. Furthermore, scytophycins were identified from Anabaena cf. cylindrica PH133, Nostoc sp. UHCC 0501 (HAN 11/1), and Scytonema sp. UHCC 0502 (HAN 3/2). In total, seven previously unidentified scytophycin variants were described from these strains. The detected activity is in line with previous reports of antifungal and cytotoxic activity found from scytophycin variants (Patterson & Carmeli 1992; Smith et al. 1993). Previously, scytophycins had been associated with terrestrial cyanobacteria (Jung et al. 1991; Tomsickova et al. 2014), but these four cyanobacterial strains were isolated from brackish or fresh water. In addition, it was the first time scytophycin production was described from a cyanobacterium belonging to the genus Anabaena. Thus, the distribution of scytophycins is clearly wider than previously thought. Compounds structurally similar or related to scytophycins include aplyronine A from the sea hare Aplysia kurodai (Yamada et al. 1993), lobophorolide from the seaweed Lobo- phora variegata (Kubanek et al. 2003), ulapualide and kabiramide from nudibranch eggmasses (Matsunaga et al. 1986; Roesener & Scheuer 1986), and the sponge-associated compounds halichondramide (Kernan & Faulkner 1987), mycalolide (Hori et al. 1993), sphinxolide (Guella et al. 1989), and swinholide (Carmeli & Kashman 1985; Kobayashi et al. 1989). During study II, three cyanobacterial strains (Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110, and Nostoc sp. UHCC 0450 [N107.3]) were found to display antifungal activity but their active compounds were not identified. Nostoc sp. UHCC 0450 was active against A. flavus. Subsequently, further purification and analysis with spectrometric methods revealed that Nostoc sp. UHCC 0450 produces cytotoxic swinholides (III). Therefore, the antifungal screening revealed potent natural products with cytotoxic activity (scytophycin, swinholide, and hassallidin). These findings led to the discovery of the putative biosynthesis genes for swinholide (III) and verification of the biosynthesis genes for the production of scytophycins (III), which had just been described (Ueoka et al. 2015). Further activity tests were not carried out with scytophycins or swinholides since both of these macrolide natural products have been described to be cytotoxic actin-targeting agents against several cell lines (Carmeli & Kashman 1985; Kobayashi et al. 1990; Patterson & Carmeli 1992; Smith et al. 1993; Bubb et al. 1995). The hassallidin producing strains Anabaena spp. UHCC 0688 (BIR JV1) and UHCC 0780 (HAN 7/1), and Nostoc spp. CENA 219 and 6 sf Calc where either active against C. albicans, or both C. albicans and A. flavus. Differences in activity were most likely due to the different amounts of hassallidin produced by the strains. Together the four strains produce more than nine variants of hassallidins. Some of these variants are new but their exact structures were not described during this study. Previously, hassallidin A and B have shown antifungal activity against Cryptococcus neoformans, Aspergillus spp., Fusarium spp., Penicillium sp., Ustilago maydis and Acremonium strictum (Neuhof et al. 2005, 2006a,b). Hassallidins, including balticidins (Bui et al. 2014), have been known mainly for their antifungal activity. However, hassallidins A and B have also shown activity against human acute T-cell leukemia cells (Jurkat ATCC-TIB-152) and murine aneuploid fibrosarcoma cells (L 929) (Neuhof et al.

27 2006a). Nostoc sp. UHCC 0306 (HIID D1.B) was found to be active in antileukemic screening and was shown to produce hassallidin (I). Its aqueous extract showed potent activity against MOLM-13 cells but also against hepatocytes. The causal connection between hassallidin production and cytotoxic activity was not verified but this led us to suspect that hassallidins show wide cytotoxic activities. In study IV, hassallidin D was purified from Anabaena sp. UHCC 0258 (previously Ana- baena sp. 258). The structure of the main variant (4,6-diacetylmannose-hassallidin D) was elucidated with MS and NMR analyses, and the previously uncertain configurations of sug- ars were identified (-D-mannose, -D-arabinose, N-acetyl--D-glucosamine) (Figure 4). Four variants of hassallidin D with varying amounts or positions of acetyl groups were purified, and they showed IC50 values ranging from 2.6 to 4.0 μM against MOLM-13 and NRK cell lines. Hassallidin variants also lysed freshly isolated mouse hepatocytes. Previously, di-

AcMan-hassallidin D (from Anabaena sp. SYKE 748A) has shown IC50 values ranging from 0.29 to 1.0 μM against different Candida strains (Vestola et al. 2014). Differences between the results may be explained by methodological differences. Our further analysis revealed that mammalian cells were more susceptible against hassallidin D. Propidium iodide (PI) staining confirmed the lytic activity of hassallidin D, and the estimated half-maximal effective concentration (EC50) was 4.8 μM against MOLM-13 cells and 30 μM against C. albicans with 15 minute incubation of hassallidin D.

Figure 4. Structure of 4,6-diacetylmannose-hassallidin D, the main variant produced by Anabaena sp. UHCC 0258.

28 The results confirmed that hassallidins, especially hassallidin D in this case, are cytotoxic compounds. Structurally identical or similar compounds have been described also from other bacteria showing antifungal activity. These compounds include the herbicolins (Aydin et al. 1985), jagaricin (Graupner et al. 2012), chromobactomycin (Kim et al. 2014), and Sch 20561 and 20562 (Afonso et al. 1999a,b). They also show very similar antifungal activities without antibacterial activity.

4.1.3 Culture collections and screening (I–IV) As stated, cyanobacteria are a prolific source of natural products (Burja et al. 2001; Singh et al. 2011; Gerwick & Moore 2012). The antileukemic and antifungal screening studies here continued this work of finding activities and proceeding to find novel natural products from cyanobacteria (I, II). Screening studies are still an important part of discovering natural products, and culture collections are valuable sources of strains for these. The UHCC culture collection consists of axenic, i.e. pure, cyanobacterial strains and non-axenic strains. The purification of a cyanobacterium into an axenic culture is time-consuming and difficult due to the close association between cyanobacteria and other bacteria. The importance of main- taining culture collections was highlighted in this study as several new active cyanobacterial strains were found and possibly novel compound candidates were detected (I, II). In addition, the culture collection played a crucial part in this thesis, as hassallidin producers had been identified previously (Vestola et al. 2014) and swinholide and scytophycin producing strains were also found among the cyanobacterial strains in the UHCC culture collection (II, III, IV). Although the utility of culture collections should not be underestimated, they are also associated with certain problems. For the production of hassallidin, an efficient producer strain was screened for but one of the best candidates displayed decreased production during mass cultivation. The disappearance of hassallidin production has also been observed before (Wang et al. 2012). Currently, culture collections are criticized for artificial conditions and loss of functions seen in field samples, but this study showed that cyanobacteria can produce bioactive compounds even after decades in the laboratory. For instance, the oldest laboratory strain in this study was isolated in 1984 (Planktothrix agardhii UHCC 0127 from lake Vesijärvi, Finland), and it showed anti-AML activity. Other studies have also demonstrated that cultured cyanobacteria maintain their bioactivities for decades (Oftedal et al. 2011). During study I, activities were also confirmed by repeating the cytotoxicity assay with extracts collected in different batch cultures, which did not affect the activity levels of extracts. The use of axenic cultures proved its value in study III, where we could demonstrate that the terrestrial cyanobacterium Nostoc sp. UHCC 0450 produces swinholide A. Previ- ously, swinholides have been discovered mostly in association with marine sponges, especially the species Theonella (Kobayashi et al. 1989, 1990; Kitagawa et al. 1990; Tsukamoto et al. 1991; Youssef & Mooberry 2006). In addition, an uncultivated heterotrophic bacterial symbiont of Theonella sp. has been identified to produce a swinholide variant (Ueoka et al.

29 2015). Thus, two disparate species produce almost the exact same compound. However, the origin of the biosynthesis genes remains unclear: Evidence for horizontal gene transfer was found, but we were unable to identify the original source of the genes (III). Previously, swinholide production had been associated only once with cyanobacteria when field samples of Symploca cf. sp. and Geitlerinema sp. were described to contain swinholide and ankaraholides (Andrianasolo et al. 2005). However, nine new swinholide variants, named samholides, were recently described from a field collection of the cyanobacterium cf. Phor- midium sp. collected from the American Samoa (Tao et al. 2018). Thus, it is evident that cyanobacteria produce swinholide-type compounds, including scytophycins, which were already from the start identified from cyanobacteria (Moore et al. 1986). Studies I and II were carried out with crude extracts while in study IV a purified compound was used to carry out cytotoxicity assays. This has a major impact on the interpreta- tion of the results. With crude extracts, the activity might be caused by multiple compounds together that are maybe even produced by multiple organisms as not all of the cyanobacterial strains studied here were axenic. The study with a purified compound produced clearer results regarding the active compound, in this case hassallidin D (IV).

4.2 Discovering biosynthesis pathways (III) Cyanobacterial natural products are produced often by PKS, NRPS, or hybrid pathways. The swinholide biosynthesis genes were identified from the Nostoc sp. UHCC 0450 draft genome, which has a size of 7.32 Mb. The swinholide biosynthesis gene cluster (swi) is a trans- AT PKS and encodes five PKS proteins (SwiC to SwiG) of which one is a standalone AT (SwiG) (Figure 5). The swi cluster had typical features of a trans-AT PKS, with non-elongating domains and split modules. In addition, the cluster included the pyran synthase for ring formation, another aberrant DH putatively involved in the formation of another ring, and tan- dem ACPs. The identification of the biosynthesis gene cluster was based on the previous discovery of the biosynthesis gene cluster for the swinholide variant misakinolide from the heterotrophic symbiont bacterium Candidatus “Entotheonella serta” TSWA-1 (Ueoka et al. 2015). Swinholide and misakinolide vary only by two double bond carbon-hydrogen units, thereby causing misakinolide to be a smaller compound by 52 Da. Swinholide and misakinolide biosynthesis gene cluster proteins have an almost identical set of catalytic domains (Figure 6) but in swinholide biosynthesis all modules are used while in the misakinolide cluster the last module is skipped (Ueoka et al. 2015). The biosynthesis gene cluster for scytophycin was found from the 5.74 Mb draft genome of Anabaena sp. UHCC 0451. Previously Ueoka et al. (2015) had described the biosynthesis gene cluster for one of the scytophycin variants, tolytoxin (6-hydroxy-7-OMe-scytophycin-B) from Scytonema sp. PCC 10023. The main variant of Anabaena sp. UHCC 0451 was 7-OMe-scytophycin-B, and it may be assumed that the tto and scp clusters represent the pathway for all scytophycin variants, which span at least 34 compounds (see Supplementary Material in study II). The biosynthesis gene clusters for scytophycin synthesis (tto and scp) from two different cyanobacteria were almost identical for four main proteins (ScpC to

30 Figure 5. Genes and pathway of putative 85-kb swinholide biosynthesis gene cluster. AT, acyltransferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthase, KS0, nonelongating KS; MT, methyltransferase; OMT, O-methyltransferase; PS, pyransynthase; TE, thioesterase; •, acyl carrier protein.

ScpF), and standalone AT (ScpA), as well as even additional ORFs surrounding the clusters were similar (ScpB, ScpG) (Figure 6). The similarity (identity) between scp and tto amino acid sequences was 78 to 83% (BLASTp), which was comparable to swinholide and misakinolide similarity (75 to 84%). In addition, another tolytoxin/scytophycin cluster was described from Planktothrix sp. PCC 11201 (Pancrace et al. 2017a), which was called tto2 in study III. Overall, only a few trans-AT clusters have been described from cyanobacteria, including the swinholide biosynthesis cluster, the three tolytoxin/scytophycin biosynthesis clusters, and the luminaolide biosynthesis gene cluster from Planktothrix paucivesiculata PCC 9631 (Ueoka et al. 2015; Pancrace et al. 2017a). These biosynthesis pathways together with the misakinolide pathway formed their own cluster in a phylogenetic analysis of trans-AT proteins and differed from other bacterial trans-AT biosynthesis pathways such as that for the cyanobacterial compound nosperin (Kampa et al. 2013). Recently, the number of cyanobacterial pathways has increased, for instance, through the identification of the phormidolide trans-AT PKS (Bertin et al. 2016). However, to perform better evolutionary analy-

31 sis, more trans-AT pathways similar to swi and mis are needed, as evidenced by our horizontal gene transfer analysis. HGT events were seen in several PKS modules of swinholide, misakinolide, luminaolide, and scytophycin pathways but a common donor for the genes could not be found. The donor suggestions from the HGTector analysis were surprisingly diverse. Small putative transposase ORFs are often found near large biosynthesis clusters, which was the case also here. This supports the horizontal gene transfer hypothesis but the close resemblance between all PKS modules and the commonly occurring repetition in PKS and NRPS genes complicate the evolutionary analysis. The highly repetitive NRPS and PKS genes may cause also problems in genome assembly, and often additional sequencing is needed to assemble the full biosynthesis gene clusters. However, a large size makes it easier to find a cluster, and thus the swinholide and scytophycin biosynthesis gene clusters could be found quite easily from the draft genomes as the structures of the compounds were known. During the thesis, another approach to find natural products was initiated (unpublished data). Some of the cyanobacteria exhibiting antileukemic activity in study I were chosen for further analysis where genomic DNA was purified, sequenced, and assembled into draft genomes. These draft genomes were scanned for NRPS and PKS genes manually and with antiSMASH (Medema et al. 2011). The amount of discovered pathways varied between strains but overall these draft genomes harbored many NRPS and PKS, as expected (Table 9). Because of lack of information regarding the structures of active compounds, determining pathways for further investigation was not possible and the work needs to be continued. Identifying biosynthesis pathways can be a stepping stone to finding other strains producing identical or similar compounds and to modifying the production or the compound. For instance, the NRPS pathway for hassallidin was initially described from Dolichospremum sp. UHCC 0090 (Anabaena sp. 90) (Wang et al. 2012; Vestola et al. 2014) following the identification of other producer cyanobacteria based on the presence of has genes (Vestola et al. 2014; Pancrace et al. 2017 [V]; Abreu et al. 2018).

32 , 0 ) found from found ) tto (1) ) from swi sp. PCC 11201 (*, gene n. Planktothrix sp. UHCC 0451, tolytoxin ( ) from tto (2) Anabaena Swinholide biosynthesis gene cluster ( cluster gene biosynthesis Swinholide ) from PCC 9631, and second tolytoxin ( se; PS, pyransynthase; TE, thioesterase; •, acyl carrier protei T, acyltransferase; DH, dehydratase; KS, ketosynthase, KS ER, enoyl reductase; KR, ketoreductase; Entotheonella” sp. TSWA-1, scytophycin ( scp -AT PKS biosynthesis gene clusters (domain level). (domain clusters gene biosynthesis PKS -AT trans Planktothrix paucivesiculata Candidatus ) from “ mis sp. PCC 10023, luminaolide ( lum ) from sp. UHCC 0450, misakinolide ( Figure 6. Comparison of six related swinholide-type related six of Comparison 6. Figure Nostoc for PL11201360002 sequence is located in a different contig). A nonelongating KS; MT, methyltransferase; OMT, O-methyltransfera Scytonema

33 2017). Cluster numbers are etails) and assembled into draft 8 19 6 13 1 10 9 21 5 10 34 14 18 4 15 1 9 75 21 20 et al. clusters of compounds such as lassopep- 1 0 1 3 1 1 4 2 3 4 4 et al. 2011; Blin 5 4 3 6 3 6 6 5 1 3 5 Some of the or showing cyanobacterial antileukemic strains Terpene Bacteriocin Others Total nobacterial strains used in study I. 69 1 0 0 439 3 1 1 669 0 2 1 107 3 0 2 827 1 1 1 888862 1 1 2178 2513 1 2 1 1 0 1 1 3 218205 4 3 0 1 3 2 5.5 8.5 6.5 7.0 9.5 8.2 11.8 18.6 17.7 12.9 18.1 Draft genome size (Mb) Scaffolds NRPS PKS NRPS-PKS UHCC 0127 sp. UHCC 0262 sp. UHCC 0290 sp. UHCC 0266 sp. UHCC 0305 sp. UHCC 0276 sp. UHCC 0263 sp. UHCC 0301 sp. UHCC 0187 Cyanobacteria Anabaena Anabaena Geminocystis Geminocystis Leptolyngbya Nostoc sp. UHCC 0302 Nostoc sp. UHCC 0306 Phormidium Planktothrix agardhii Pseudanabaena Trichormus preliminary results and probably include false clusters (e.g. partial clusters). The group “others” includes the biosynthesis The group “others” includes clusters (e.g. partial clusters). and probably include false preliminary results and indoles. tides, microviridins, cyanobactins Table 9. Number of biosynthesis clusters detected from some cya cytotoxic activity were sequenced with the Illumina HiSeq2500 p latform (Macrogen, 300–500 bp library; see study III for more d genomes. The nucleotide sequences were scanned for biosynthesis clusters with antiSMASH 4.2.0 (Medema 34 4.3 Elucidating mechanism of action (IV)

Describing a natural product raises questions about the purpose and function of the compound. Describing the mechanism of action is essential especially for pharmaceutical evaluation. Unfortunately, most studies stop after chemical characterization of the compound, and cyanobacteria are no exception. There are plenty of cyanobacterial natural products waiting for their mechanism of action to be elucidated. Nevertheless, cyanobacterial natural products have a wide variety of MOAs (Salvador-Reyes & Luesch 2015). For instance, there are many microfilament-targeting compounds, including both actin- and tubulin-targeting agents (Salvador-Reyes & Luesch 2015). Examples of actin-targeting compounds include the swinholides (Bubb et al. 1995) and scytophycins (Smith et al. 1993). In study IV, the MOA for hassallidins was described. Hassallidin D, which was purified from Anabaena sp. UHCC 0258, was found to be a cytotoxic compound against different eukaryotic cells (IV), enabling the study of the MOA with mammalian cells. Ultrastructural analysis and visualization of cell damage turned out to be highly useful for determining the MOA. Morphological changes in transmission electron microscopy samples showed clearly that the cell surface membranes of MOLM-13 cells were disrupted (Figure 7). Starting by disappearance of the microvilli and continuing into several micrometer long disruptions in the surface membrane, the cells finally lysed completely. The lytic activity of hassallidin D resembled the activity of another cyanobacterial lipopeptide, anabaenolysin (Jokela et al. 2012; Oftedal et al. 2012). In addition, similar to anabaenolysin A (Oftedal et al. 2012), hassallidin D was found to be a more potent membrane disrupter than digitonin. Digitonin and hassallidin behaved similarly in cell assays, with both, for instance, affecting especially cholesterol containing membranes and leaving the mitochondria unharmed. The MOA of hassallidin D is, however, unlikely to be the same as the MOA of digitonin. Our in silico analysis showed rapid intercalation of hassallidin D into cholesterol containing membranes but not specific attachment to cholesterol (Figure 8), which has, in contrast, been described for digitonin (Nishikawa et al. 1984; Frenkel et al. 2014; Sudji et al. 2015). Interestingly, the results indicated that cholesterol created a more clearly structured membrane where hassallidin D was able to bind. The cytotoxicity analysis of four purified hassallidin D variants indicated that the activity of hassallidin D was independent of the number and position of acetyl groups in the hassallidin D mannose unit. However, the ring structure of hassallidin may be essential for directing the fatty acid into the membrane, as it has been shown that the ring structure opens easily and results in decreased activity (Vestola et al. 2014). During in silico modeling, tyrosine-5 stayed close to the cholesterol membrane and the conformation of the hassallidin molecule was more stable (due to invading lipid tail) than with a membrane without cholesterol. More studies are needed to comprehensively understand how hassallidin interacts with the membrane at the molecular level. Moreover, some natural products have multiple MOAs, such as the antifungal drug amphotericin B, which may create a channel through the membrane but also binds directly to ergosterol and disturbs membrane stability (Gray et al. 2012).

35 individual . The TEM images A to D show the ultrastructural morphology of n cell membranes Figure 7. Hassallidin D induces cell lysis and ruptures mammalia MOLM-13 cells, a control or a cell treated with hassallidin D. Images E to H show morphology in detail, with red mi, mitochondria. endoplasmic reticulum; arrows n, nucleus; er, mv, microvilli; marked in images: indicatingsurface membrane. Organelles broken or discontinuous cell

36 The MOA of hassallidin could also be more specific than indicated. For instance, the antibiotic drug daptomycin interacts with cell membrane phospholipids and especially phospha- tidylglycerol, which may explain its specificity against bacterial cells (Taylor & Palmer 2016). However, fully describing the mechanism of action of a compound can be very difficult. For instance, the mechanism of action of even daptomycin is still not completely understood. The lack of antibacterial activity in hassallidins and other identical or similar compounds (see Table 4) was hypothesized to be caused by their cholesterol-dependency, which makes hassallidins cytotoxic against eukaryotic cells containing sterols. The only exception for antibacterial activity in the hassallidin family is the anti-mycobacterial activity of herbicolin A (Freundt & Winkelmann 1984), although unlike other bacteria, mycobacteria do contain sterols. In study IV, it was shown that hassallidin D disrupts yeast and mammalian cell surface membranes. A previous study with Candida albicans also suggested that in fungi or yeast the cell surface membrane is affected (Neuhof et al. 2006c). Furthermore, here hassallidin D lysed mammalian cells faster than yeast cells, which was indicated to be due to the physical barrier, cell wall, in yeasts. Thus, it might be that the MOA is similar for all eukaryotic cells. However, it could be argued that cyanobacteria are more likely to encoun- ter fungi and small animals than large mammals in their habitats where they could compete with or defend themselves against these. The reasons why cyanobacteria produce hassallidin or any other natural product are mainly speculative, and very little is known with high confidence. Hassallidins are, nevertheless, produced by many cyanobacterial strains across the world (Vestola et al. 2014; Abreu et al. 2018), which may indicate that detergent type compounds are important for cyanobacteria. In addition, other bacteria produce similar detergent-type compounds. Especially Bacillus spp. produce many detergent compounds such as the lipopeptides iturins (Maget-Dana et al. 1985; Cochrane & Vederas 2016). These compounds affect bacterial hydrophobicity and thereby the adhesion of bacteria to different surfaces (Ahimou et al. 2000). As evidenced by this study on hassallidin D, cell assays can provide valuable clues regarding the mechanism of action of a compound. Especially microscopic methods provide rapid insights into cell death type (necrotic, apoptotic, etc.). Thus, the cell assays in study I could already have shown clues regarding the MOA of these samples if the samples had been inspected more closely. However, microscopy is problematic with cyanobacterial crude extracts since color and other physical features hinder the analysis.

37 Figure 8. Images captured from in silico modeling video of hassallidin D and POPC/cholesterol (A) or POPC bilayers (B).

38 5 Conclusions and future prospects

This thesis demonstrates that cyanobacteria continue to be a prolific source of bioactive natural products. Activity-guided research of new natural products resulted in the identification of novel producer strains of known natural products from new habitats and potential activity hits, which could indicate new natural products. This study increased the knowledge regarding antileukemic and antifungal activities found from cyanobacteria, especially expanding the knowledge regarding the known cytotoxic compounds hassallidins, swinholides, and scytophycins. The biosynthesis gene clusters for swinholide from Nostoc sp. and scytophycin from Anabaena sp. were determined. Furthermore, a mechanism of action for hassallidin was proposed. Screening studies against desired bioactivity represent a valid research strategy as novel compounds are still found from various environments (Pye et al. 2017). It is accepted that cyanobacteria still harbor many unknown natural products; however, the amount of these is uncertain. Here we found several strains with possibly novel compounds. Further- more, as shown in this study, the prediction of bioactive hits based on sampling location, habitat type or genus is difficult. However, it must also be acknowledged that some among these newly found compounds may later be identified to be known natural products, which are unfamiliar to our identification system. The rediscovery of known compounds is inevi- table and there is a pressing need for effective identification strategies. Molecular networking, for instance, is trying to answer this need for de-replication (Nguyen et al. 2013b; Wang et al. 2016). Turning a natural product into a useful product for the pharmaceutical or another sec- tor of industry is a very long process. The discovery of a compound is the first step. Deter- mining bioactivities, biosynthesis and mechanisms of action, such as in this study, are the next steps, which provide clues regarding the level of toxicity and specificity of the compound. The antifungal compound hassallidin was revealed to have broad lytic activity, mak- ing it a low-interest compound, for instance, for the treatment of fungal diseases. However, natural products commonly have many unfavorable features (toxicity, specificity, solubility, production level, etc.) that have to be improved before a compound finds use. One of the bottlenecks in this process is the large-scale production of the active compound as it is usually a minor component within the complex matrix of all the compounds produced by the organism. The amount of a bioactive compound needed, for example, in the drug development process rapidly increases along the process from a few micrograms to hundreds of grams. Several approaches have been adopted to overcome supply problems such as synthetic chemistry or genetic engineering. The identification of the swinholide and scytophycin biosynthesis gene clusters makes it possible to modify these natural products genetically and to modify their production to solve supply problems. Single enzymes from biosynthesis pathways may also become useful in combinatorial biosynthesis approaches (binding of natural biosynthesis and synthetic methods) (Winn et al. 2016). Overall, only a small fraction

39 of natural products becomes useful but all identified compounds expand the chemical space of compounds regardless of the activity. Natural product research is also connected to ecological and evolutionary aspects of the organisms. In this study, we showed evidence for horizontal gene transfer of the scytophycin and swinholide biosynthesis pathways. The origins of the genes remain unknown but the findings support the concept that bacteria frequently share these clusters. In addition, hassallidins are surprisingly widespread compounds among cyanobacteria as well as other bacteria. Why these natural products are found from diverse places and organisms and why they are produced remain open questions. For instance, hassallidin can be hypothesized to act as a defense agent against fungi but may just as well be similar to Bacillus lipopeptides that are involved in the attachment of bacteria to surfaces (Ahimou et al. 2000). The knowledge produced in this thesis can serve as a starting point for further investigations regarding the function of hassallidins. Importantly, in the future, the specificity of hassallidin toward fungal ergosterol compounds should be compared to the activity tested here against cholesterol membranes. In study III, whole genome sequencing was used to identify natural product biosynthesis gene clusters. Some of the strains from study I were also sequenced. The genomic approach is thriving in natural product research, and it will continue to be important in the future. It shows high promise for natural product discovery as novel compounds are found even among silent or cryptic biosynthesis clusters. With a tremendous increase in the amount of data, better algorithms are also needed to process it. This does not apply to the analysis of genomic data alone but generally to computational approaches (for instance, in silico analyses and molecular networking). Importantly, after several years of dwindling of natural product discovery, the field is now more popular than ever. Cyanobacteria play a key role in this, as shown by the studies in this thesis: Cyanobacteria remain an underex- plored group of organisms that harbor many natural products about which we know very little.

40 6 Acknowledgements

This work was carried out at the Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Finland. Part of the study was conducted at the Centre for Pharmacy, Department of Clinical Science, Faculty of Medicine, University of Bergen, Nor- way. Doctoral candidate funding was provided by the Doctoral Programme in Microbiology and Biotechnology, University of Helsinki. First of all, I wish to thank my supervisor Professor Kaarina Sivonen for the opportunity to successfully carry out this thesis in the Cyano Group and for all the scientific advice and mentoring during these years. I thank my second supervisor Professor Lars Herfindal for the great opportunity to visit the lab in Bergen several times. Actually, I may not have even undertaken this thesis project if it was not for the first visit to your lab, so I thank you for all the shared enthusiasm, interest, and support over the years. I thank my third supervisor Docent Jouni Jokela for always being open-minded in discussions. I greatly appreciate our discussions on various topics regarding science and life. For pre-examining my thesis, I thank Professor Lena Gerwick and Docent Päivi Tam- mela. I thank Professor Jeanette H. Andersen for agreeing to act as my opponent. I am grateful to all co-authors: You made the publications possible. I wish to thank present and former members of the Cyano Group and the technical personnel at the Department, especially Matti and Lyudmila. I am also grateful to my other “research family” at the University of Bergen: Thank you to the group, and Brith and other technicians. Special thanks go to my dear friends far away: Ronja, Sarah and Tara! I would like to gratefully acknowledge my friends in field; I mean Tuulia, Iina, Dina, Johannes and Heikki. Thank you for sharing the ups and downs! And yet another thanks to Johannes for language revision. Finally, I thank my dear family and close friends for all the support you always show me. Thank you, Lauri, for bearing with me during the stressful times and for all the help you have given with, simply, everything. Lastly, I want to commemorate my aunt and grandma, who showed that life is for living!

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55 Supplemental Table 1. Cyanobacteria strains used in the thesis. The listed factors include the genus, new UHCC coding and other strain names, Ax = axenicity, geographical origin, growth habitat, year of isolation, the accession number of 16S rRNA sequence when available, and the article where the strain was used (an empty field indicates that the data is not available).

Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article Anabaena sp. UHCC 0175 XPORK 1D - Porkkala cape, FIN benthic 1999 I UHCC 0181 XPORK 6B - Porkkala cape, FIN benthic 1999 AJ630414 I, II UHCC 0182 XPORK 6C - Porkkala cape, FIN benthic 1999 EF568904 I UHCC 0187 XPORK 11B - Porkkala cape, FIN benthic 1999 I UHCC 0220 XPORK 35A - Porkkala cape, FIN benthic 1999 EF583855 I UHCC 0224 XPORK 36C - Porkkala cape, FIN benthic 1999 I UHCC 0225 XPORK 36D - Porkkala cape, FIN benthic 1999 EF583853 I UHCC 0228 XPORK 4D - Porkkala cape, FIN benthic 1999 I UHCC 0229 XPORK 5C - Porkkala cape, FIN benthic 1999 I UHCC 0230 XPORK 13A - Porkkala cape, FIN benthic 1999 I UHCC 0233 XSPORK 7B - Porkkala cape, FIN benthic 1999 KF631399 I UHCC 0245 XSPORK 27B - Porkkala cape, FIN benthic 1999 I UHCC 0248 XSPORK 36B - Porkkala cape, FIN benthic 1999 EF583861 I UHCC 0253 XSPORK 2A + Porkkala cape, FIN benthic 1999 EF583854 I UHCC 0257 XSPORK 27C - Porkkala cape, FIN benthic 1999 I UHCC 0258 258 - Lake Tuusulanjärvi, FIN 1992 IV UHCC 0261 XSPORK 14D + Porkkala cape, FIN benthic 1999 I UHCC 0272 XPORK 15D + Porkkala cape, FIN benthic 1999 EF583860 I UHCC 0274 XSPORK 7A - Porkkala cape, FIN benthic 1999 I UHCC 0301 HIID D7.A - Hiidenvesi, Kiihtelyksenselkä, FIN sediment 1999 + I, II UHCC 0309 XHIID B6 - Hiidenvesi, Mustionselkä sediment 1999 II UHCC 0315 315 + Coast of Helsinki, FIN planktonic 1997 I UHCC 0318 318 + Coast of Helsinki, FIN planktonic 1998 I UHCC 0334 XHIID B2.A - Hiidenvesi, Mustionselkä, FIN sediment 1999 II UHCC 0335 XHIID B5 - Hiidenvesi, Mustionselkä, FIN sediment 1999 + I UHCC 0348 XPORK 15F - Porkkala cape, FIN benthic 1999 I UHCC 0350 XPORK 1C - Porkkala cape, FIN benthic 1999 I UHCC 0355 XPORK 2A - Porkkala cape, FIN benthic 1999 I UHCC 0362 XPORK 6A - Porkkala cape, FIN benthic 1999 I UHCC 0451 HAN 21/1 + Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, III UHCC 0506 HAN 37/1 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0688 BIR JV1 - The Gulf of Finland + II UHCC 0780 HAN 7/1 - Kobben, Hanko, FIN benthic 2012 II UHCC 0815 HAN 15/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0821 HAN 15/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II ATCC 29413 Mississippi, USA planktonic 1964 NR_074300 II CENA 247 Rio Camarão, CE, Brazil planktonic 2009 II PCC 7108 Moss Beach, California USA 1970 AJ133162 II PCC 7122 Cambridge, UK planktonic 1939 AF091150 II PH 133 Lake Arresø, DK 1993 AJ293110 II XPORK 15A Porkkala cape, FIN benthic 1999 I XPORK 16A Porkkala cape, FIN benthic 1999 I XPORK 2E Porkkala cape, FIN benthic 1999 I XPORK 34A Porkkala cape, FIN benthic 1999 EF583863 I

56 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article Aphanocapsa sp. CENA 223 Morro Branco, CE, Brazil 2009 II Aphanothece sp. UHCC 0316 XHIID D1 - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II Calothrix sp. UHCC 0022 - Baltic Sea, FIN 2009 II UHCC 0166 XPORK 1A - Porkkala cape, FIN benthic 1999 I UHCC 0192 XPORK 20A - Porkkala cape, FIN benthic 1999 I UHCC 0221 XPORK 36A - Porkkala cape, FIN benthic 1999 I UHCC 0234 XSPORK 4A - Porkkala cape, FIN benthic 1999 I UHCC 0235 XSPORK 3 - Porkkala cape, FIN benthic 1999 I UHCC 0273 XSPORK 10A - Porkkala cape, FIN benthic 1999 I UHCC 0336 Cal 336/3 + Lake Enäjärvi, FIN planktonic 1999 II UHCC 0363 XPORK 9A - Porkkala cape, FIN benthic 1999 AM230670 I UHCC 0366 Cal 336/2 - Lake Enäjärvi, FIN planktonic 1999 II UHCC 0503 HAN 24/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I UHCC 0504 HAN 33/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0505 HAN 38/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I UHCC 0781 HAN 19/2 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0782 HAN 30/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0784 HAN 33/1 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0785 HAN 20/3 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0786 HAN 22/2 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0787 HAN 21/5 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0788 HAN 6/4 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0789 HAN 3/1a - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I UHCC 0790 HAN 22/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0791 HAN 21/4 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0792 HAN 17/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I, II UHCC 0793 HAN 20/2 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I UHCC 0798 HAN 37/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0800 HAN 37/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0802 HAN 30/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + II UHCC 0812 HAN 8/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I UHCC 0813 HAN 16/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + I UHCC 0818 HAN 8/2 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0822 HAN 26/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I UHCC 0823 HAN 6/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0825 HAN 16/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0826 HAN 36/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0828 HAN 21/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II UHCC 0829 HAN 38/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + I, II HAN 12/2 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II CENA 283 Piauí, Brazil terrestrial 2009 II PCC 7102 Antofagasta, Chile terrestrial 1958 II PCC 7103 Algal herbarium specimen, USA AM230700 II PCC 7507 Vierwaldstättersee, Switzerland terrestrial 1972 NR_102891 II Cyanobium sp. UHCC 0277 HIID A3.A - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 II

57 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article Cyanospira sp. CENA 215 Morro Branco, CE, Brazil 2009 II Cyanothece sp. UHCC 0190 XPORK 13B - Porkkala cape, FIN benthic 1999 I XPORK 15E Porkkala cape, FIN benthic 1999 I Cylindrospermopsis raciborskii CS-505 Freshwater, Solomon Dam, Q, Australia planktonic 1996 EU552055 II Cylindrospermum sp. PCC 7417 Stockholm, Sweden terrestrial 1972 NR_114701 II XHIID C12 Hiidenvesi, Nummelanselkä, FIN sediment 1999 II Fischerella sp. CENA 298 Paraíba, Brazil terrestrial 2009 II PCC 9339 AB075984 II Geitlerinema sp. CENA 224 Morro Branco, CE, Brazil 2009 II CENA 228 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 252 Sodrelândia, BA, Brazil planktonic 2009 II Geminocystis sp. UHCC 0266 HIID B11.B + Hiidenvesi, Mustionselkä, FIN sediment 1999 +I, II UHCC 0292 HIID A17.B - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 + I UHCC 0305 HIID B4.B - Hiidenvesi, Mustionselkä, FIN sediment 1999+ I Gloeotrichia sp. CENA 250 Rio Camarão, CE, Brazil planktonic 2009 II CENA 272 Paulista, PA, Brazil planktonic 2009 II CENA 285 Piauí, Brazil terrestrial 2009 II CENA 288 Ceará, Brazil terrestrial 2009 II Leptolyngbya sp. UHCC 0267 HIID B15.A + Hiidenvesi, Mustionselkä, FIN sediment 1999 +I, II UHCC 0287 HIID A16.A - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 + I, II UHCC 0290 HIID D2.A - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 + I, II UHCC 0293 HIID B21.B - Hiidenvesi, Mustionselkä, FIN sediment 1999 +I UHCC 0295 HIID A25.A - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 + I, II UHCC 0380 HIID A19.B - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 II CENA 229 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 254 Sodrelândia, BA, Brazil planktonic 2009 II CENA 256 Sodrelândia, BA, Brazil planktonic 2009 II CENA 282 Piauí, Brazil terrestrial 2009 II CENA 292 Ceará, Brazil terrestrial 2009 II CENA 299 Rio Grande do Norte, Brazil terrestrial 2009 II Limnothrix sp. CENA 217 Morro Branco, CE, Brazil 2009 II Merismopedia sp. CENA 264 Icó, CE, Brazil 2009 II Microchaete sp. CENA 251 Rio Camarão, CE, Brazil planktonic 2009 II Microcystis sp. Izancya 5 Lake Mira, Portugal planktonic KC311962 I (LEGE 91339)

58 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article Izancya 31 River Douro, Portugal planktonic KC311961 I (LEGE 91347) Izancya 42 Lake Bracas, Portugal planktonic KC311967 I (LEGE 91352) Izancya 1 Lake Mira, Portugal planktonic I (LEGE 91093) Izancya 7 Lake Vela, Portugal planktonic I Izancya 30 River Vilar, Portugal planktonic I Izancya 43 Lake Bracas, Portugal planktonic I (LEGE 91353) Myxosarcina sp. CENA240 Cisterna, Canindé, CE, Brazil 2009 II CENA241 Cisterna, Canindé, CE, Brazil 2009 II CENA242 Cisterna, Canindé, CE, Brazil 2009 II CENA244 Cisterna, Canindé, CE, Brazil 2009 II Nostoc sp. UHCC 0152 N 152 + Lake Sääksjärvi, FIN planktonic 1986 AJ133161 II UHCC 0159 N 159 + Lake Haukkajärvi, FIN planktonic 1986 II UHCC 0178 XPORK 4A - Porkkala cape, FIN benthic 1999 I UHCC 0194 XPORK 22A - Porkkala cape, FIN benthic 1999 + I UHCC 0195 XPORK 24A - Porkkala cape, FIN benthic 1999 I UHCC 0196 XPORK 24B - Porkkala cape, FIN benthic 1999 I UHCC 0222 UK 222IIC - Mikkeli, FIN symbiotic + I UHCC 0251 XPORK 5A + Porkkala cape, FIN benthic 1999 I, II UHCC 0252 XPORK 14A + Porkkala cape, FIN benthic 1999 I UHCC 0255 XSPORK 13A + Porkkala cape, FIN benthic 1999 I UHCC 0270 XHIID A6 + Hiidenvesi, Mustionselkä, FIN sediment 1999 II UHCC 0302 XHIID C2 + Hiidenvesi, Nummelanselkä, FIN sediment 1999 + I, II UHCC 0306 HIID D1.B + Hiidenvesi, Kiihtelyksenselkä, FIN sediment 1999 + I UHCC 0308 XHIID A1 - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 II UHCC 0314 XHIID D14 - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II UHCC 0317 XHIID C3 - Hiidenvesi, Nummelanselkä, FIN sediment 1999 II UHCC 0319 XHIID C4 - Hiidenvesi, Nummelanselkä, FIN sediment 1999 II UHCC 0322 XHIID C5A - Hiidenvesi, Nummelanselkä, FIN sediment 1999 II UHCC 0324 XHIID D13 - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II UHCC 0325 XHIID D12 - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II UHCC 0333 XHIID D7 + Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II UHCC 0341 HIID C18.A - Hiidenvesi, Nummelanselkä, FIN sediment 1999+I, II UHCC 0342 XHIID C5B - Hiidenvesi, Nummelanselkä, FIN sediment 1999 II UHCC 0345 XHIID D8 - Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 II UHCC 0347 XPORK 15C - Porkkala cape, FIN benthic 1999 I UHCC 0360 UK S60 II + Scotland, UK symbiotic + I UHCC 0450 N 107.3 + Lichen-associated, FIN symbiotic 1990 KP701040 II, III UHCC 0501 HAN 11/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0503 HAN 24/1 - Kobben, Hanko, Baltic sea, FIN epilithic 2012 +II UHCC 0611 IO-102 I - Sysmä, FIN epilithic II UHCC 0689 BIR LS7 - Gulf of Finland II UHCC 0692 UK 1 - Oulunkylä, Helsinki, FIN symbiotic 2006 II UHCC 0694 UK 4 - Itä-Pakila, Helsinki, FIN symbiotic 2006 II UHCC 0697 UK 18aI - Autti, FIN symbiotic 2006 + II UHCC 0698 UK 18aIII - Autti, FIN symbiotic 2006 II

59 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article UHCC 0699 UK 18aIV - Autti, FIN symbiotic 2006 II UHCC 0744 UK 18bI - Autti, FIN symbiotic 2006 II UHCC 0745 UK 18bII - Autti, FIN symbiotic 2006 II UHCC 0746 UK 18bIII - Autti, FIN symbiotic 2006 II UHCC 0747 UK 18bIV - Autti, FIN symbiotic 2006 II UHCC 0768 UK 220I b - Mikkeli, FIN symbiotic + I UHCC 0769 UK 104IIa - Teeri-Lososuo, Kuhmo, FIN symbiotic + I UHCC 0770 UK 89IIa Hitonhaudan rotko, FIN symbiotic + I UHCC 0771 UK 222Ib - Mikkeli, FIN symbiotic + I UHCC 0772 UK 2aImI - Helsinki, FIN symbiotic + I UHCC 0773 UK S81.I - Scotland, UK symbiotic + I UHCC 0776 UK 35 - FIN symbiotic II UHCC 0777 UK 92Ic - Hitonhaudan rotko, FIN symbiotic + I UHCC 0786 HAN 22 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0797 HAN 27 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 + II UHCC 0814 HAN 10/1 - Kobben, Hanko, Baltic sea, FIN benthic 2012 + II UHCC 0834 N 135.9.1 - FIN symbiotic + I, II UHCC 0910 N 134.1 Lichen-associated, FIN symbiotic + I UHCC 0923 N 138 Lichen-associated, FIN symbiotic + I CENA 219 Morro Branco, CE, Brazil 2009 + II CENA 221 Morro Branco, CE, Brazil 2009 II CENA 227 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 234 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 237 Lagoa Povoado Nova Aurora, CE, Brazil planktonic 2009 II CENA 238 Lagoa Povoado Nova Aurora, CE, Brazil planktonic 2009 II CENA 239 Lagoa Povoado Nova Aurora, CE, Brazil planktonic 2009 II CENA 257 Sodrelândia, BA, Brazil planktonic 2009 II CENA 258 Sodrelândia, BA, Brazil planktonic 2009 II CENA 259 Sodrelândia, BA, Brazil planktonic 2009 II CENA 261 Sodrelândia, BA, Brazil planktonic 2009 II CENA 269 Paulista, PA, Brazil planktonic 2009 II CENA 271 Paulista, PA, Brazil planktonic 2009 II CENA 274 Bahia, Brazil terrestrial 2009 II CENA 275 Bahia, Brazil terrestrial 2009 II CENA 277 Bahia, Brazil terrestrial 2009 II CENA 278 Bahia, Brazil terrestrial 2009 II CENA 293 Ceará, Brazil terrestrial 2009 II CENA 296 Ceará, Brazil terrestrial 2009 II N 115.3.2 - FIN symbiotic II N 122.4.B - FIN symbiotic II N 123.4 - FIN symbiotic II N 124.3.B - FIN symbiotic II N 990622III - FIN symbiotic II N 990653.17 - FIN symbiotic II UK 7bIIm - Itä-Pakila, Helsinki, FIN symbiotic 2006 II XHIID C1 Hiidenvesi, Nummelanselkä, FIN sediment 1999 II PCC 7120 NR_074310 II PCC 73102 Australia terrestrial 1973 AF027655 II 6 (sf. Calc) Dobre Pole, Czech Republic 1998 II 113.5 - FIN symbiotic + I

60 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article 116.6.23 - FIN symbiotic II 116.7.5 - FIN symbiotic II Phormidium sp. UHCC 0002 2 + Lake Markusbölefjärden, FIN planktonic 1985 AJ133185 I UHCC 0214 214 - Östra Kyrksundet, Åland, FIN planktonic 1987 + I UHCC 0218 218 - Bodomjärvi; Espoo, FIN planktonic 1987 + I UHCC 0276 HIID B22.A - Hiidenvesi, Mustionselkä, FIN sediment 1999 +I CENA 270 Paulista, PA, Brazil planktonic 2009 JQ771627 II HIID B22.B Hiidenvesi, Mustionselkä, FIN sediment 1999 II Planktothrix sp. UHCC 0018 18 + Lake Långsjön, Åland, FIN planktonic 1985 + I UHCC 0036 245 - Lappalanjärvi, FIN planktonic 1989 + I UHCC 0045 45 + Lake Enäjärvi, Vihti, FIN planktonic 1985 + I UHCC 0126 126/3 + Lake Tuusulanjärvi, FIN planktonic 1986 AJ133166 I UHCC 0127 127 + Lake Vesijärvi, Lahti, FIN planktonic 1984 AJ133168 I UHCC 0216 289 - Baltic sea, FIN planktonic 1992 + I UHCC 0226 226 - FIN planktonic + I UHCC 0278 278 - Rasio, Naantali waterworks, FIN planktonic 1991 + I UHCC 0280 251 + Dragsfjärd, FIN planktonic 1989 + I CENA 236 Povoado Novo Aurora, CE, Brazil planktonic 2009 II Pseudanabaena sp. UHCC 0262 HIID B16.A + Hiidenvesi, Mustionselkä, FIN sediment 1999 +I UHCC 0289 HIID A13.A - Hiidenvesi, Kirkkojärvi, FIN sediment 1999 + I, II UHCC 0294 HIID B19 - Hiidenvesi, Mustionselkä, FIN sediment 1999 + I, II UHCC 0297 HIID C15.A - Hiidenvesi, Nummelanselkä, FIN sediment 1999II CENA 235 Povoado Novo Aurora, CE, Brazil planktonic 2009 II CENA 266 Paulista, PA, Brazil planktonic 2009 II CENA 267 Paulista, PA, Brazil planktonic 2009 II CENA 287 Piauí, Brazil terrestrial 2009 II Rivularia sp. UHCC 0191 XPORK 16B - Porkkala cape, FIN benthic 1999 I UHCC 0351 XPORK 22B - Porkkala cape, FIN benthic 1999 + I Scytonema sp. UHCC 0321 XPORK 15B - Porkkala cape, FIN benthic 1999 + I UHCC 0502 HAN 3/2 - Kobben, Hanko, Finland benthic 2012 II CENA 268 Paulista, PA, Brazil planktonic 2009 II PCC 7110 Bermuda epilithic 1971 II Stigonema sp. CENA 291 Ceará, Brazil terrestrial 2009 II CENA 295 Ceará, Brazil terrestrial 2009 II Synechococcus sp. CENA 222 Morro Branco, CE, Brazil 2009 II CENA 232 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 233 Açude São Mateus, CE, Brazil planktonic 2009 II CENA 249 Rio Camarão, CE, Brazil planktonic 2009 II CENA 253 Sodrelândia, BA, Brazil planktonic 2009 II Tolypothrix sp. UHCC 0227 XPORK 34B - Porkkala cape, FIN benthic 1999 II UHCC 0328 Tol 328 - Kuopio, Finland terrestrial 1999 II

61 Growth Year of 16S rRNA UHCC code Other code Ax Geographical origin habitat isolation sequence Article UHCC 0690 BIR MGR4 - Gulf of Finland II UHCC 0822 HAN 26/2 - Hanko Casino sea shore, Hanko, Finland benthic 2012 + II HAN 5/1 - Kobben, Hanko, Finland epilithic 2012 + II HAN 25/1 - Kobben, Hanko, Finland epilithic 2013 + II PCC 7101 Borneo terrestrial 1950 II PCC 7415 Stockholm, Sweden terrestrial 1972 II PCC 7504 Stockholm, Sweden benthic 1972 II Trichormus sp. UHCC 0263 HIID D3 + Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 + I, II UHCC 0250 HIID D4 + Hiidenvesi, Kiihkelyksenselkä, FIN sediment 1999 + I UHCC 0264 HIID B6.A + Hiidenvesi, Mustionselkä, FIN sediment 1999II Unidentified UHCC 0778 HAN 20/1 - Kobben, Hanko, FIN benthic 2012 II UHCC 0783 HAN 19/3 - Kobben, Hanko, FIN benthic 2012 II UHCC 0796 HAN 29/3 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 II UHCC 0799 HAN 3/1 - Kobben, Hanko, FIN benthic 2012 II UHCC 0803 HAN 40/1 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 II UHCC 0811 HAN 21/2 - Kobben, Hanko, FIN benthic 2012 II UHCC 0817 HAN 6/1 - Kobben, Hanko, FIN benthic 2012 II UHCC 0819 HAN 32/2 - Hanko Casino sea shore, Baltic Sea, FIN benthic 2012 II UHCC 0827 HAN 15/3 - Kobben, Hanko, FIN epilithic 2012 II CENA 263 Baixão dos Bois, BA, Brazil 2009 II CENA 276 Bahia, Brazil terrestrial 2009 II CENA 281 Piauí, Brazil terrestrial 2009 II CENA 294 Ceará, Brazil terrestrial 2009 II HAN 31/a - Kobben, Hanko, FIN epilithic 2012 II