Simultaneous Production of Anabaenopeptins and Namalides by the

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Simultaneous production of anabaenopetins and namalides by the cyanobacterium Nostoc sp. CENA543 Tania K. Shishido, Jouni Jokela, David P Fewer, Matti Wahlsten, Marli F. Fiore, and Kaarina Sivonen ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00570 • Publication Date (Web): 21 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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ACS Chemical Biology

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Simultaneous production of anabaenopetins and namalides by the cyanobacterium

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Nostoc sp. CENA543

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Tânia K. Shishido1, Jouni Jokela1, David P. Fewer1, Matti Wahlsten1, Marli F. Fiore2, Kaarina

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Sivonen1*

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Department of Food and Environmental Sciences, University of Helsinki, Viikki Biocenter 1, P.O.

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Box 56, 00014 University of Helsinki, Finland. 2Center for Nuclear Energy in Agriculture,

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University of São Paulo, Avenida Centenário 303, Piracicaba, 13400-970, São Paulo, Brazil.

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Corresponding Author

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*Email: [email protected].

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1 ACS Paragon Plus Environment


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ABSTRACT

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Anabaenopeptins are a diverse group of cyclic peptides, which contain an unusual ureido linkage.

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Namalides are shorter structural homologs of anabaenopeptins, which also contain an ureido

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linkage. The biosynthetic origins of namalides are unknown despite a strong resemblance to

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anabaenopeptins. Here we show the cyanobacterium Nostoc sp. CENA543 strain producing new

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(nostamide B–E (2, 4, 5 and 6)) and known variants of anabaenopeptins (schizopeptin 791 (1) and

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anabaenopeptin 807 (3)). Surprisingly, Nostoc sp. CENA543 also produced namalide B (8) in

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similar amounts as anabaenopeptins, and the new namalides D (7), E (9) and F (10). Analysis of the

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complete Nostoc sp. CENA543 genome sequence indicates that both anabaenopeptins and

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namalides are produced by the same biosynthetic pathway through module skipping during

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biosynthesis. This unique process involves the skipping of two modules present in different

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nonribosomal peptide synthetases during the namalide biosynthesis. This skipping is an efficient

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mechanism since both anabaenopeptins and namalides are synthesized in equally significant

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amounts by Nostoc sp. CENA543. Consequently, gene skipping may be used to increase and

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possibly broaden the chemical diversity of related peptides produced by a single biosynthetic gene

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cluster. Genome mining demonstrated that the anabaenopeptin gene clusters are widespread in

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cyanobacteria and can also be found in tectomicrobia bacteria.

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INTRODUCTION

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Anabaenopeptins are cyclic hexapeptides that contain a D-lysine in the ring, an N-methylated

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fifth amino acid, a side chain amino acid connected through its α-amino group via an ureido bond to

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the D-Lys α-amino group and a C-terminal amino acid closing the macrocyclic ring to the D-Lys δ-

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amino group (Figure 1). Anabaenopeptins frequently contain non-proteinogenic amino acids

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including homotyrosine (Hty) and homophenylalanine (Hph) (Supplementary Table S1). Many

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anabaenopeptins are protease and phosphatase inhibitors of carboxypeptidase A, protein

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phosphatase 1, metallo carboxypeptidase TAFIa (thrombin activatable fibrinolysis inhibitor),

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trypsin and chymotrypsin, while others show weak or no bioactivities in the tests performed1–4.

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Anabaenopeptins were recently discovered to be potent inhibitors of blood clot stabilizing

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carboxypeptidases that have been found to be an alternative to anticoagulants, which are the most

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prescribed drugs3,4. Some anabaenopeptin variants specifically inhibit metallo carboxypeptidase

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TAFIa, an important target involved in the blood coagulation cascade, in high potency with IC50

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values of 2.1 and 1.5 nM3,4.

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Anabaenopeptins are the products of a nonribosomal peptide synthetase (NRPS) biosynthetic

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pathways (aptABCD) encoded in the genomes of a variety of cyanobacteria5–8. The anabaenopeptin

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biosynthetic pathway deviates from the NRPS colinear rule, in which the order and number of

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modules dictates the number and position of amino acids in the final chemical structure of the

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peptide6. The anabaenopeptin gene cluster from Anabaena sp. 90 encodes two alternative loading

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modules, which allow the simultaneous synthesis of multiple variants of anabaenopeptins6.

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Additional exceptions to the biosynthetic logic of nonribosomal peptide assembly have also been

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reported in the literature including module skipping or module iteration9,10. Tailoring enzymes are

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frequently involved in the biosynthesis of non-proteinogenic amino acids11. Homo-amino acids are

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non-proteinogenic amino acids that contain a methylene (-CH2-) group in the carbon side chain and

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HphABCD enzymes were described to be involved in the synthesis of homophenylalanine and



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possibly homotyrosine12. The hphABCD genes were located around the anabaenopeptin gene cluster

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of Nostoc punctiforme PCC 7310212 and Sphaerospermopsis torques-reginae ITEP-0248.

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Anabaenopeptins are widely distributed in cyanobacterial genera6,13. There are 115

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anabaenopeptin variants differing in amino acid composition of which, 104 originate from

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cyanobacteria, 8 from theonellid sponge, one from sponge Psammocinia and 2 variants were found

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from oyster2,4,14–18 (Supplementary Table S1). Anabaenopeptins have diverse names, such as

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nodulapeptin,

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pompanopeptin, schizopeptin, keramamides, konbamide, mozamides, paltolides and psymbamide

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depending of the organism/source isolated (Supplementary Table S1).

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brunsvicamide,

Namalides are

ferintoic

acid,

lyngbyaureidamide,

nostamide,

oscillamide,

cyclic tetrapeptides, which bear striking structural similarity to

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anabaenopeptins but lack two amino acids from the macrocycle19,20 (Figure 1). Namalide is a

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carboxypeptidase A inhibitor at submicromolar level (IC50 of 250 ± 30 nM) and have been reported

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from the marine sponge Siliquariaspongia mirabilis19. New namalide variants have recently been

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also reported from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-02420. However,

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the biosynthetic origin of namalide and the relationship between namalide and anabaenopeptins is

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unclear.

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Here we report the simultaneous production of anabaenopeptins and namalides by the

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cyanobacterium Nostoc sp. CENA543, isolated from a Brazilian saline-alkaline lake (Nhecolândia,

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Pantanal). The complete genome obtained from this strain contains biosynthetic gene cluster for

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anabaenopeptin but lacks a separate and specific biosynthetic pathway for namalide, which suggests

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that namalide is a module skipping product from the anabaenopeptin biosynthetic pathway.

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RESULTS AND DISCUSSION

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Anabaenopeptin and namalide from Nostoc sp. CENA543


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Anabaenopeptins are protease and phosphatase inhibitors that are almost exclusively reported

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from cyanobacteria or environmental samples containing cyanobacteria (Supplementary Table S1).

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HPLC-ITMS and UPLC-QTOF analysis indicated that Nostoc sp. CENA543, isolated from a

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saline-alkaline lake in Nhecolândia (Pantanal wetland area in Brazil), produces two structurally

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homologous compound groups, anabaenopeptins and namalides (Figure 1 and Table 1). These

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compounds eluted after the earlier reported nodularins and pseudospumigins21 (Figure 2). A

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methanol extract of the culture was first analyzed with HPLC-ITMS leading to the identification of

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anabaenopeptins 1–3 and 5 (Supplementary Figures S1–S2). HPLC-ITMS analysis identified

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anabaenopeptins with four different ion masses, m/z 778, 792, 806 and 808 [M+H]+. UPLC-QTOF

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analysis demonstrated that there are six anabaenopeptin variants, three of which have m/z 778 and

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present different amino acids in positions one, three and five (Table 1, Supplementary Figures S3–

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S4). Ion assignments of the high resolution product ion spectra verified the anabaenopeptins

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chemical structures (Supplementary Table S2 and Figure S5). The side chain amino acid was

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predicted to be Ile (with 100% score) based on analysis of the AptA_Ad1 adenylation domain

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binding pocket (Supplementary Table S3). The AptB adenylation domain binding pocket prediction

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(position three) had a score of 70% and predicted to select isoleucine. This low score may indicate

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that leucine can be incorporated as well. However, leucine is less frequent in position 3 than

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isoleucine (Table 2). Anabaenopeptin 1 was earlier published as schizopeptin (Sp) 791 from

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Schizothrix sp.22 and anabaenopeptin 3 as anabaenopeptin 807 from Nodularia spumigena23. The

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other four variants (2, 4, 5 and 6) with chemical structures Ile/Val-CO-cyclo[Lys-Ile/Val-Hph-

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MeAla/Ala-Hph/Phe] are, to our knowledge, new and were named nostamide B – E since nostamide

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A has been earlier reported from Nostoc punctiforme PCC731026.

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Surprisingly Nostoc sp. CENA543 was also found to produce namalide (Figure 1). HPLC-

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ITMS product ion spectra of protonated namalide D (7) is substantially different from the

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anabaenopeptin spectra (Supplementary Figure S2), which prevented the straightforward



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recognition of the namalide (Supplementary Figure S6). Comparison of the UPLC-QTOF product

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ion spectra of protonated namalide B from Sphaerospermopsis torques-reginae ITEP-024 to spectra

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from cyanobacterium Nostoc sp. CENA543 yielded a perfect fit (Figure 3). Namalide B and C

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structures from Sphaerospermopsis torques-reginae ITEP-024 have been obtained using MS, NMR

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and amino acid analysis20 proving the presence of namalides also in Nostoc sp. CENA543. New

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namalide variants, based on the UPLC-QTOF product ion spectra, were detected in the Nostoc sp.

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CENA543 extract (Supplementary Figures S7 and S8, Supplementary Table S4). Namalide was

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first reported from the marine sponge Siliquariaspongia mirabilis19. The structure of this namalide

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is Phe-CO-cyclo[Lys-Ile-Phe] and has a different compliment of amino acids with the exception of

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Lys compared to namalides detected from the cyanobacteria Nostoc sp. CENA543 and

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Sphaerospermopsis torques-reginae ITEP-02420 (Table 1).

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A shared anabaenopeptin and namalide biosynthetic gene cluster

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The biosynthetic origins of namalide are unclear. We obtained a complete genome sequence

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from Nostoc sp. CENA543 to identify the biosynthetic pathways involved in the synthesis of

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anabaenopeptin and namalides. The 7.2 Mb Nostoc sp. CENA543 genome has a GC content of

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40.84 % and consists of a single 6.99 Mb chromosome and five plasmids ranging in size from 30–

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67 kb (Figure 4). A Prokka genome automatic annotation predicted 10 rRNAs, 6042 CDS, 15 repeat

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regions, 1 tmRNA and 75 tRNA.

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A prediction of the Nostoc sp. CENA543 secondary metabolite gene repertoire based on the

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complete genome sequence indicated the presence of 20 possible biosynthetic gene clusters, six of

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which contained nonribosomal peptide synthetase genes (Supplementary Table S5). Two of these

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biosynthetic gene clusters have been recently assigned to be involved in the synthesis of nodularins

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and pseudospumigins21. Two biosynthetic pathways are hybrid NRPS/PKS (polyketide synthase)

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gene clusters and they could result in compounds with six (cluster 4) and three (cluster 14) amino


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acids plus malonyl-CoA units if they are not silent (Figure 4 and Supplementary Table S5). A fifth

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possible nonribosomal biosynthetic gene cluster contains just one module with one adenylation

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domain. The anabaenopeptin gene cluster is 26 kb and contains four NRPS (AptA, AptB, AptC and

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AptD) and an ABC transporter (AptE), with 2-isopropylmalate synthase (HphA) and an ORF

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(NTF2-like) genes encoded between apt genes (Supplementary Tables S5 and S6). The biosynthesis

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of namalide would require a biosynthetic pathway containing four adenylation domains. However,

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no such nonribosomal peptide synthetase biosynthetic gene cluster containing four adenylation

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domains with suitable substrate prediction based on their binding pocket was identified from the

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genome sequence (Supplementary Table S5). The only plausible biosynthetic gene cluster involved

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in the synthesis of namalides is the anabaenopeptin biosynthetic gene cluster (Supplementary Table

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S5). The predictions of the amino acids incorporated by the adenylation domain are also in

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accordance with the chemical structure of both compounds (Figure 1 and Supplementary Table S7).

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This analysis strongly suggests that namalide is produced by a module skipping event, between the

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second domain of AptC and the condensation-adenylation domains of AptD, during the synthesis of

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anabaenopeptins (Figure 5A).

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The module-skipping process has been previously reported in the synthesis of myxochromide

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S due the presence of an inactive mutated peptidyl carrier protein domain (PCP) in myxobacteria24.

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Another mechanism for module skipping was described in the combinatorial engineering of

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polyketide synthase and involves ACP (acyl carrier protein)-to-ACP chain transfer25. Small 11- and

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14-residues peptaibol peptides are synthesized by NRPS from Trichoderma fungi, in which three

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modules may be skipped26. The module skipping in the cyanobacteria Nostoc sp. CENA543 is

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unique in the fact that the second module of AptC (condensation-adenylation-thiolation domains)

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and partial module of AptD (condensation and adenylation domains) are skipped but the

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thioesterase from AptD might still be used for the cyclization and release of the namalide. The

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alignments of AptC and AptD sequences from producers of namalides and anabaenopeptins



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(CENA543 and ITEP-024) and sequences from other strains that produce only anabaenopeptins,

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indicate that these sequences are mostly similar with the exception of a gap in the CENA543 and

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ITEP-024 condensation domain sequences from AptD (Supplementary Figure S9–S11). Further

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biochemical analysis will be necessary to characterize the enzymes involved in the

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anabaenopeptin/namalide synthesis and to test this hypothesis.

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Mining of anabaenopeptin gene clusters and possible namalide producers

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Anabaenopeptins are produced by a broad range of cyanobacteria, while namalides has been

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detected only in a marine sponge19, the cyanobacteria Sphaerospermopsis torques-reginae ITEP-

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02420 and Nostoc sp. CENA543 (this study). Anabaenopeptins and namalides have a D-lysine

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connected with an ureido bond to a side chain amino acid. The first adenylation domain of AptA is

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responsible for the selection and incorporation of this lysine and thus this region is a conserved

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marker to detect anabaenopeptin producers through gene sequence comparison. Here we searched

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for truncated anabaenopeptins biosynthetic gene clusters that could be involved in the synthesis of

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anabaenopeptins and namalides. Anabaenopeptin gene clusters were detected in 56 genomes (out of

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568 cyanobacterial genomes analyzed) belonging to diverse genera of cyanobacteria but also in the

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genome of the tectomicrobia Candidatus Entotheonella sp. TSY1 (Supplementary Figure S12). The

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anabaenopeptin biosynthetic gene clusters are spread throughout the cyanobacterial phylum (Figure

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6). Twenty five cyanobacterial strains that contain anabaenopeptin gene clusters were analyzed for

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namalide synthesis, but only Nostoc sp. CENA543 and Sphaerospermopsis torques-reginae ITEP-

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024 produce namalides in detectable amounts (Supplementary Table S7). No truncated

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anabaenopeptin gene clusters that would correspond to namalide gene cluster were observed.

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All of the anabaenopeptin gene clusters encoded four nonribosomal peptide genes (aptA,

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aptB, aptC, aptD) and an ABC-transporter (aptE), with few exceptions (e.g Nostoc sp. 268 lacks

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aptE and Nodularia spumigena 309 has aptD and hphA fused in one gene) (Figure 5A). The vast


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majority of which contained one aptA (82%), but ten cyanobacterial (Anabaena, Aphanizomenon

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and Nostoc genera) and the Candidatus Entotheonella sp. TSY1 encoded two alternative aptA

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(aptA1 and aptA2) genes (Figure 5B and Supplementary Figure S12). The non-colinearity of the

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anabaenopeptin synthesis has been previously discovered for Anabaena strains6. Anabaena sp. 90

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has two starter modules with adenylation domains that have substrate specificity and produce the

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different anabaenopeptins containing respectively Arg/Lys and Tyr in position one6. This is a

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mechanism used by Anabaena strains to increase the chemical diversity of the peptides produced.

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However, Planktothrix strains produce diverse anabaenopeptin variants due the promiscuity of

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adenylation domains7.

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Strains belonging to the Chroococcales, Oscillatoriales, Nostocales, and Stigonematales

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orders of cyanobacteria encoded anabaenopeptin biosynthetic pathways that varied from 24.7 kb to

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33.6 kb. The nomenclature describing the anabaenopeptins and the anabaenopeptins biosynthetic

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gene clusters vary. In the case of the anabaenopeptin gene clusters, there is a variation according to

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the strain, such as ana or apn for Planktothrix5,7, apt for Anabaena, Microcystis, Nodularia, Nostoc

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and Sphaerospermopsis6,8,27, or kon for the Candidatus Entotheonella28). However, just apt from

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Anabaena sp. 90 and apn from Planktothrix agardhii NIVA-CYA 126/8 are deposited in the

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Minimum Information about a Biosynthetic Gene Cluster (MIBiG)29 repository.

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Genes involved in the homo-amino acids synthesis (hphA, hphB, hphCD) were present in all

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the anabaenopeptin gene clusters, with the exception of Scytonema hofmannii PCC 7110 and

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Candidatus Entotheonella sp. TSY1 (Figure 5B). Most of the anabaenopeptin gene clusters

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contained genes involved in the homo-amino acids synthesis upstream and/or downstream the apt

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genes (Supplementary Figure S12). Homo-amino acids (homotyrosine or homophenylalanine) may

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be found in all positions of anabaenopeptins except for positions two and three (Table 2 and Figure

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5). Anabaenopeptins often contain homo-amino acids in their chemical structure and from the 115

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anabaenopeptins that have been previously described, 47 contain one, 52 contain two, one contains



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three while just 15 do not contain amino acids with methylene group elongated side chains (in

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homo-amino acids one extra methylene group is present) (Supplementary Table S1). Four of those

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15 anabaenopeptins that do not contain amino acid with methylene group are characterized from

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two cyanobacteria, and the rest are from an oyster and marine sponges (Supplementary Table S1).

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Open reading frames (ORFs) with unknown functions may be present within the

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anabaenopeptin gene clusters (Supplementary Figure S12). Two ORF insertions were found in

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between the konbamide NRPS gene cluster and the authors argued that these insertions may have

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resulted in the lack of konbamide synthesis by Candidatus Entotheonella sp. TSY128. Our analysis

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suggests that one ORF insertion in the anabaenopeptin gene cluster are common and does not

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prevent the compound synthesis, e.g. in Anabaena sp. BIR260, Nostoc spp. CENA543, N135.9.1,

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and XPORK14A, Phormidium sp. DVL1003c, Sphaerospermopsis torques-reginae ITEP-024.

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However, no anabaenopeptins were detected in the extracts of Nostoc sp. HIID D1B and Nostoc

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calcicola FACHB-389, which have two ORFs inserted between anabaenopeptin genes. Further

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analyses are necessary to unveil if these ORFs could have a role in the anabaenopeptin biosynthesis.

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Anabaenopeptin gene clusters were located close to another NRPS (0.08–17.5 kb) or hybrid

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NRPS/PKS (6.9–9.5 kb) gene clusters (50%) and/or microviridin genes (42%, 0.2–4.7 kb). This

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“meta peptide synthesis gene cluster” has been previously described for Planktothrix spp. in

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cyanobacteria.5,30 Our results demonstrate that this arrangement includes other genera such as

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Aphanizomenon, Nodularia and Oscillatoria spp. Other cyanobacterial genera, such as

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Sphaerospermopsis8, Fischerella, Nostoc and Phormidium presented one of the NRPS or

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microviridin gene cluster close to apt genes (Supplementary Figure S12). Interestingly, these NRPS

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or microviridin gene clusters situated in the same region than anabaenopeptin gene cluster are

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mostly involved in the synthesis of other protease inhibitors, such as spumigin, microginin and

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microviridin. Spumigin A is known to inhibit porcine trypsin31, thrombin, and plasmin (IC50 of 4.6,

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4.9 and 16.1 µg/mL)32, microginin inhibits angiotensin-converting enzyme (IC50 of 7.0 µg/mL)33


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and microviridin J inhibits porcine trypsin (IC50 of 0.034, 0.096 and 0.150 mg/mL for respectively,

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10, 30 and 50 mg/mL of porcine trysin), bovine chymotrypsin (IC50 of 2.80 mg/mL) and daphnid

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trypsin-like proteases (IC50 of 0.0039 mg/mL)34.

241 242

Evolutionary history of AptA

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Anabaenopeptins variants differs in the exocyclic amino acid due two different previously

244

described mechanisms: the presence of two alternative loading modules (AptA1 and AptA2) from

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Anabaena spp.

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domain sequences of the ApnA (ApnA_Ad1) from Planktothrix spp.7. The evolutionary history of

247

ApnA_Ad1, based on diverse Planktothrix strains sequences, showed four different genotypes

248

grouped according to the amino acid incorporated by this adenylation domain30. Here we observed a

249

similar pattern in the evolutionary history of full AptA sequences from diverse cyanobacteria

250

(Supplementary Figure S13). A phylogenetic tree based on AptA sequences indicates that strong

251

bootstrap supported clades are formed by sequences that have chemically similar amino acid

252

selection by the first adenylation domain (Ad1) (Supplementary Figure S13). The second

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adenylation domain (Ad2) of AptA is involved in the selection and incorporation of a lysine, which

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is detected in all anabaenopeptins previously described (Table 2). Lysine has been reported to be

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intrinsic for the improvement in the carboxipeptidase A and B inhibition for the anabaenopeptin

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brunsvicamide35. More recently, anabaenopeptins containing the positively charged amino acids

257

arginine and lysine in the exocyclic amino acid were found to be more potent metallo

258

carboxipeptidase TAFIa inhibitors4. Interestingly, cyanobacteria containing a second alternative

259

starter (AptA1 and AptA2) have AptA1 that is predicted to incorporate lysine or arginine

260

(Supplementary Figure S13) and therefore, synthesize a more potent protease inhibitor variant.

6

or due promiscuity caused by point mutations occurred in the first adenylation

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We compared the predictions and the amino acids present in the major variants of

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anabaenopeptins by combining literature review and chemical analysis (UPLC-QTOF) performed in



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this study (Supplementary Table S7). These predictions did not always agree with the chemical

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compound detected. Most of the mismatches were present in the first adenylation domain of AptC,

265

which incorporates a homo-amino acid. Scytonema hofmannii PCC 7110 did not contained homo-

266

amino acid genes close to the anabaenopeptin gene cluster and in fact did not produced

267

anabaenopeptin containing homo-amino acids (Supplementary Table S7).

268 269 270

CONCLUSIONS

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This study demonstrates that anabaenopeptins biosynthetic pathways are broadly dispersed among

272

cyanobacteria. The anabaenopeptin gene cluster was also present in one tectomicrobia bacterium

273

and although nearly all the detected anabaenopeptins in the literature are produced by cyanobacteria

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or cyanobacteria-containing organisms, there is a potential for this gene cluster to be found in other

275

bacteria. Furthermore, the high genetic diversity of anabaenopeptin gene clusters reflects the large

276

amount of chemical diversity reported and the even higher amount of anabaenopeptin variants that

277

could still be unknown. The results from this study also suggest that namalide is the product of a

278

module skipping event during the biosynthesis of anabaenopeptins.

279 280

METHODS

281

Strains and cultivation

282

Nostoc sp. CENA543 was isolated from a water sample collected in September 3, 2010 from the

283

saline-alkaline lake “Salina 67 Mil” (19°27´42″S, 56°08´21”W) located at Centenário farm in the

284

southern part of the sub-region Nhecolândia situated in the north of the municipality of Aquidauana,

285

Mato Grosso do Sul State, Brazil36. Nostoc sp. CENA543 was purified into axenic culture before

286

chemical analysis and DNA isolation. The strain was cultivated at 20–22 °C under continuous low

287

photon irradiance (5-10 µE m−2 s−1), low salinity (0.6 ‰) and high phosphorus (5500 µg PO4-P L-1)


Page 13 of 35

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Z8 medium37 without nitrogen source. Nostoc sp. CENA543 was also cultivated in 1 % salinity

289

(addition of 8.75 g NaCl L-1 and 3.75 g MgSO4·7H2O L-1) Z8 medium without nitrogen source to

290

decrease the slime formation21 prior to DNA isolation. The strains analyzed for the synthesis of

291

anabaenopeptins and/or namalides were grown in Z8 medium with or without nitrogen source under

292

the previously described conditions21. Sphaerospermopsis torques-reginae ITEP-024 was cultivated

293

as previously described8.

294 295

DNA extraction, genome sequencing and assembly

296

The genomic DNA of Nostoc sp. CENA543 was isolated as previously described21. DNA was

297

checked using a NanoDrop 1000 spectrophotometer (Thermo Scientific) to measure the

298

concentration and an Agilent TapeStation (Agilent Technologies) to assess the quality. High-

299

molecular DNA was subjected to library (Illumina TruSeq® PCR Free 350bp) construction and

300

sequenced by Illumina HiSeq2500 platform with a paired ends 100 cycles run. The genomic DNA

301

was in addition sequenced by PacBio RS II (Pacific Biosciences) to obtain long reads and to

302

complete the genome sequence. The genome was assembled using HGAP3 (SMRT Analysis 2.3).

303 304

Genome mining and in silico analysis

305

Amino acid sequences of AptA were used for the genome mining of anabaenopeptin gene clusters

306

using tBLASTn tool against the National Center for Biotechnology Information (NCBI) database

307

and a library of unpublished 67 partial cyanobacterial genomes from the University of Helsinki. The

308

genome sequence obtained from Nostoc sp. CENA543 was annotated using Prokka38 in the Galaxy

309

web server and RAST39–41. In addition, the genomes were analyzed for biosynthetic genes using

310

antiSMASH42–44 and annotated using Artemis45. The sequence was analyzed for the NRPS/PKS

311

content using PKS/NRPS Analysis46 and the substrate prediction of the adenylation domains were

312

obtained using NRPS predictor 247,48. The phylogenetic analyses were performed in the Molecular



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Page 14 of 35

313

Evolutionary Genetics Analysis (MEGA 6.06)49. Phylogenetic tree was constructed using Neighbor-

314

joining (16S rRNA genes – K2+G, AptABCD+HphA, AptA amino acids – Poisson model +G) and

315

Maximum likelihood (16S rRNA genes – K2+G+I) methods.

316 317

LC-MS analysis

318

Cells cultivated in 40 ml of liquid cultures were collected and freeze dried. Dried biomass placed in

319

2 ml plastic tubes together with 1 ml methanol and glass beads (0.5 mm diameter glass beads,

320

Scientific Industries INC) was shaken using FastPrep cell disrupter instrument three times for 30 s

321

at a speed of 6.5 ms−1. Tubes were centrifuged 10,000× g for 5 min at room temperature.

322

Supernatants were analyzed first with low resolution HPLC-ESI-ITMS (Agilent 1100 Series

323

LC/MSD Ion Trap XCT Plus, Agilent Technologies). Ten µl sample was injected to Luna C18

324

column (2.1 x 150 mm, 5 µm, Phenomenex), which was eluted from 30 % acetonitrile (solvent B)

325

in 0.1 % HCOOH to 70 % of B (v/v) in 49 mins at 40 °C with a flow rate of 0.15 ml min-1. Mass

326

spectral data was accumulated in ultrascan positive electrospray ionization mode (26,000 m/z s-1) at

327

scan range of m/z 300 – 2200 and by averaging three spectra.

328

High resolution UPLC-QTOF analyses were performed with Acquity I–Class UPLC–Synapt G2-Si

329

HDMS (Waters Corp.) system. The first gradient program used to run from 0.1 to 1 µl of sample

330

injected to Kinetex® 1.7 µm C8 100 Å, LC column 50 x 2.1 mm, 1.6 µm (Phenomenex), consisted

331

of elution at 40 °C with a flow rate of 0.3 ml min-1 from 5 % acetonitrile/isopropanol (1:1, v/v) (+

332

0.1 % HCOOH) (solvent B) in 0.1% HCOOH to 100 % of B in 5 mins and kept there 2 mins, then

333

back to 5 % of B in 0.5 mins and finally kept there 2.5 mins before next run. In the second gradient

334

program, from 0.1 to 1 µl sample was injected to Kinetex® 1.7 µm C8 100 Å, LC Column 50 x 2.1

335

mm, Phenomenex, which was eluted at 40 °C with a flow rate of 0.3 ml min-1 from 30 %

336

acetonitrile/isopropanol (1:1) (+ 0.1 % HCOOH) (solvent B) in 0.1% HCOOH to 40 % of B in 5

337

mins and lifted to 100% in 0.01 min kept there 1.99 mins, then back to 30 % of B in 0.5 mins and


Page 15 of 35

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338

finally kept there 2.5 mins before next run (v/v). UPLC-QTOF was calibrated with sodium formate

339

and Ultramark® 1621 giving a calibrated mass range from m/z 50 - 2000. Leucine Enkephalin was

340

used at 10 s interval as a lock mass reference compound.

341 342

Accession Codes

343

Accession numbers of anabaenopeptin gene cluster (MF741679–MF741700) and 16S rRNA gene

344

(MF680040–MF680055) sequences obtained in this study are indicated in Supplementary Tables

345

S8. Accession numbers of AptA from Nostoc sp. UKS60II (MF882922) and genome sequence of

346

Nostoc sp. CENA543 (CP023278–CP023283) were also obtained in this study.

347 348

SUPPORTING INFORMATION

349

Supporting Information Available: This material is available free of charge via the Internet.

350

Literature review of anabaenopeptin variants (Supporting Information TableS1) (PDF)

351

Supporting Tables and Figures (Supporting Information) (PDF)

352 353

ACKNOWLEDGEMENTS

354

This work was supported by the grants from the Academy of Finland to K. Sivonen (1273798) and

355

D. P. Fewer (1259505) and from the São Paulo Research Foundation to M. F. Fiore (FAPESP,

356

2013/50425-8). The authors thank L. Saari for purification and cultivation of the cyanobacteria

357

strains and L. Heinilä for the DNA extraction. The authors acknowledge the support of the Freiburg

358

Galaxy Team: S. Lott and R. Backofen, Bioinformatics, University of Freiburg, Germany, funded

359

by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and

360

German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC (de.NBI)).

361

The authors would like to thank P.K. Laine and L. Paulin (Institute of Biotechnology) for the

362

assembling of the genome.



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363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Tania Keiko Shishido: 0000-0002-9156-4105 Jouni Jokela: 0000-0001-5096-3575 Matti Wahlsten: 0000-0002-4107-1695 David P. Fewer: 0000-0003-3978-4845 Marli F. Fiore: 0000-0003-2555-7967 Kaarina Sivonen: 0000-0002-2904-0458

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REFERENCES

380

1. Repka, S., Koivula, M., Harjunpä, V., Rouhiainen, L., and Sivonen, K. (2004) Effects of

381

phosphate and light on growth of and bioactive peptide production by the cyanobacterium

382

Anabaena strain 90 and its anabaenopeptilide mutant. Appl Environ Microbiol. 70, 4551-

383

4560.

384

2. Spoof, L., Błaszczyk, A., Meriluoto, J., Cegłowska, M., and Mazur-Marzec, H. (2015)

385

Structures and activity of new anabaenopeptins produced by Baltic Sea cyanobacteria. Mar

386

Drugs 14, 8.

387

3. Halland, N., Brönstrup, M., Czech, J., Czechtizky, W., Evers, A., Follmann, M., Kohlmann,

388

M., Schiell, M., Kurz, M., Schreuder, H.A., and Kallus, C. (2015) Novel small molecule

389

inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) from natural

390

product anabaenopeptin. J Med Chem. 58, 4839-4844.

391

4. Schreuder, H., Liesum, A., Lönze, P., Stump, H., Hoffmann, H., Schiell, M., Kurz, M., Toti,

392

L., Bauer, A., Kallus, C., Klemke-Jahn, C., Czech, J., Kramer, D., Enke, H., Niedermeyer,

393

T.H., Morrison, V., Kumar, V., and Brönstrup, M. (2016) Isolation, co-crystallization and

394

structure-based characterization of anabaenopeptins as highly potent inhibitors of activated

395

thrombin activatable fibrinolysis inhibitor (TAFIa). Sci Rep. 6, 32958.

396

5. Rounge, T.B., Rohrlack, T., Nederbragt, A.J., Kristensen, T., and Jakobsen, K.S. (2009) A

397

genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides

398

in a Planktothrix rubescens strain. BMC Genomics. 10, 396.

399

6. Rouhiainen, L., Jokela, J., Fewer, D.P., Urmann, M., and Sivonen, K. (2010) Two

400

alternative starter modules for the non-ribosomal biosynthesis of specific anabaenopeptin

401

variants in Anabaena (Cyanobacteria). Chem Biol. 17, 265-273.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 35

402

7. Christiansen, G., Philmus, B., Hemscheidt, T., and Kurmayer, R. (2011) Genetic variation of

403

adenylation domains of the anabaenopeptin synthesis operon and evolution of substrate

404

promiscuity. J Bacteriol. 193, 3822-3831.

405

8. Lima, S., Alvarenga, D., Etchegaray, A., Fewer, D.P., Jokela, J., Varani, A.M., Sanz, M.,

406

Dörr, F., Pinto, E., Sivonen, K., and Fiore, M.F. (2017) Genetic organization of

407

anabaenopeptin and spumigin biosynthetic gene clusters in the cyanobacterium

408

Sphaerospermopsis torques-reginae ITEP-024. ACS Chem Biol. 12, 769-778.

409

9. Amoutzias, G.D., Van de Peer, Y., and Mossialos, D. (2008) Evolution and taxonomic

410

distribution of nonribosomal peptide and polyketide synthases. Future Microbiol. 3, 361-

411

370.

412 413 414 415

10. Corre, C., and Challis, G.L. (2009) New natural product biosynthetic chemistry discovered by genome mining. Nat Prod Rep. 26, 977-986. 11. Marahiel, M.A. (2009) Working outside the protein-synthesis rules: insights into nonribosomal peptide synthesis. J Pept Sci. 15, 799-807.

416

12. Koketsu, K., Mitsuhashi, S., and Tabata, K. (2013) Identification of homophenylalanine

417

biosynthetic genes from the cyanobacterium Nostoc punctiforme PCC73102 and application

418

to its microbial production by Escherichia coli. Appl Environ Microbiol. 79, 2201-2208.

419

13. Welker, M., and von Döhren, H. (2006) Cyanobacterial peptides - nature's own

420

combinatorial biosynthesis. FEMS Microbiol Rev. 30, 530-563.

421

14. Horigome, Y., Satake, M., Oshima, Y., Yasumoto, T., and Lee, J.-S. (1999) Structure and

422

synthesis of bitter taste peptide from Korean oysters. Tennen Yuki Kagobutsu Toronkai

423

Koen Yoshishu 41, 409-414.

424 425

15. Adiv, S., and Carmeli, S. (2013) Protease inhibitors from Microcystis aeruginosa bloom material collected from the Dalton Reservoir, Israel. J Nat Prod. 76, 2307-2315.


Page 19 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


426

16. Tooming-Klunderud, A., Sogge, H., Rounge, T.B., Nederbragt, A.J., Lagesen, K., Glöckner,

427

G., Hayes, P.K., Rohrlack, T., and Jakobsen, K.S. (2013) From green to red: horizontal gene

428

transfer of the phycoerythrin gene cluster between Planktothrix strains. Appl Environ

429

Microbiol. 79, 6803-6812.

430

17. Teta, R., Della Sala, G., Glukhov, E., Gerwick, L., Gerwick, W.H., Mangoni, A., and

431

Costantino, V. (2015) Combined LC-MS/MS and molecular networking approach reveals

432

new cyanotoxins from the 2014 cyanobacterial bloom in Green Lake, Seattle. Environ Sci

433

Technol. 49, 14301-14310.

434

18. Harms, H., Kurita, K.L., Pan, L., Wahome, P.G., He, H., Kinghorn, A.D., Carter, G.T., and

435

Linington, R.G. (2016). Discovery of anabaenopeptin 679 from freshwater algal bloom

436

material: Insights into the structure-activity relationship of anabaenopeptin protease

437

inhibitors. Bioorg Med Chem Lett. 26, 4960-4965.

438

19. Cheruku, P., Plaza, A., Lauro, G., Keffer, J., Lloyd, J.R., Bifulco, G., and Bewley, C.A.

439

(2012) Discovery and synthesis of namalide reveals a new anabaenopeptin scaffold and

440

peptidase inhibitor. J Med Chem. 55, 735-742.

441

20. Sanz, M., Salinas, R.K., and Pinto, E. (2017) Namalides B and C and spumigins K-N from

442

the cultured freshwater cyanobacterium Sphaerospermopsis torques-reginae. J Nat

443

Prod.2017 (in press).

444

21. Jokela, J., Heinilä, L., Shishido, T.K., Wahlsten, M., Fewer, D.P., Fiore, M.F., Permi, P.,

445

Haapaniemi, E., and Sivonen, K. (2017) Brazilian benthic Nostoc sp. CENA543 comprises

446

hepatotoxic nodularin synthesized in high quantities and new protease inhibitor peptide

447

group pseudospumigins. Front Microbiol. (Revised manuscript ID286203).

448 449

22. Reshef, V., and Carmeli, S. (2002) Schizopeptin 791, a new anabeanopeptin-like cyclic peptide from the cyanobacterium Schizothrix sp. J Nat Prod. 65, 1187-1189.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 35

450

23. Mazur-Marzec, H., Kaczkowska, M.J., Agata Blaszczyk, A., Akcaalan, R., Spoof, L., and

451

Meriluoto, J. (2013) Diversity of peptides produced by Nodularia spumigena from various

452

geographical regions. Mar Drugs 11, 1–19.

453

24. Wenzel, S.C., Meiser, P., Binz, T.M., Mahmud, T., and Müller, R. (2006) Nonribosomal

454

peptide biosynthesis: point mutations and module skipping lead to chemical diversity.

455

Angew Chem Int Ed Engl. 45, 2296-2301.

456

25. Thomas, I., Martin, C.J., Wilkinson, C.J., Staunton, J., and Leadlay, P.F. (2002) Skipping in

457

a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer. Chem Biol. 9, 781-

458

787.

459

26. Degenkolb, T., Karimi Aghcheh, R., Dieckmann, R., Neuhof, T., Baker, S.E., Druzhinina,

460

I.S., Kubicek, C.P., Brückner, H., and von Döhren, H. (2012) The production of multiple

461

small peptaibol families by single 14-module Peptide synthetases in Trichoderma/Hypocrea.

462

Chem Biodivers. 9, 499-535.

463

27. Humbert, J.F., Barbe, V., Latifi, A., Gugger, M., Calteau, A., Coursin, T., Lajus, A.,

464

Castelli, V., Oztas, S., Samson, G., Longin, C., Medigue, C., de Marsac, N.T. (2013) A

465

tribute to disorder in the genome of the bloom-forming freshwater cyanobacterium

466

Microcystis aeruginosa. PLoS ONE 8, e70747.

467

28. Wilson, M.C., Mori, T., Rückert, C., Uria, A.R., Helf, M.J., Takada, K., Gernert, C.,

468

Steffens, U.A., Heycke, N., Schmitt, S., Rinke, C., Helfrich, E.J., Brachmann, A.O., Gurgui,

469

C., Wakimoto, T., Kracht, M., Crüsemann, M., Hentschel, U., Abe, I., Matsunaga, S.,

470

Kalinowski, J., Takeyama, H., and Piel, J. (2014) An environmental bacterial taxon with a

471

large and distinct metabolic repertoire. Nature 506, 58-62.

472

29. Medema, M.H., Kottmann, R., Yilmaz, P., Cummings, M., Biggins, J.B., Blin, K., de

473

Bruijn, I., Chooi, Y.H., Claesen, J., Coates, R.C., Cruz-Morales, P., Duddela, S., Düsterhus,

474

S., Edwards, D.J., Fewer, D.P., Garg, N., Geiger, C., Gomez-Escribano, J.P., Greule, A.,


Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


475

Hadjithomas, M., Haines, A.S., Helfrich, E.J., Hillwig, M.L., Ishida, K., Jones, A.C., Jones,

476

C.S., Jungmann, K., Kegler, C., Kim, H.U., Kötter, P., Krug, D., Masschelein, J., Melnik,

477

A.V., Mantovani, S.M., Monroe, E.A., Moore, M., Moss, N., Nützmann, H.W., Pan, G.,

478

Pati, A., Petras, D., Reen, F.J., Rosconi, F., Rui, Z., Tian, Z., Tobias, N.J., Tsunematsu, Y.,

479

Wiemann, P., Wyckoff, E., Yan, X., Yim, G., Yu, F., Xie, Y., Aigle, B., Apel, A.K.,

480

Balibar, C.J., Balskus, E.P., Barona-Gómez, F., Bechthold, A., Bode, H.B., Borriss, R.,

481

Brady, S.F., Brakhage, A.A., Caffrey, P., Cheng, Y.Q., Clardy, J., Cox, R.J., De Mot, R.,

482

Donadio, S., Donia, M.S., van der Donk, W.A., Dorrestein, P.C., Doyle, S., Driessen, A.J.,

483

Ehling-Schulz, M., Entian, K.D., Fischbach, M.A., Gerwick, L., Gerwick, W.H., Gross, H.,

484

Gust, B., Hertweck, C., Höfte, M., Jensen, S.E., Ju, J., Katz, L., Kaysser, L., Klassen, J.L.,

485

Keller, N.P., Kormanec, J., Kuipers, O.P., Kuzuyama, T., Kyrpides, N.C., Kwon, H.J.,

486

Lautru, S., Lavigne, R., Lee, C.Y., Linquan, B., Liu, X., Liu, W., Luzhetskyy, A., Mahmud,

487

T., Mast, Y., Méndez, C., Metsä-Ketelä, M., Micklefield, J., Mitchell, D.A., Moore, B.S.,

488

Moreira, L.M., Müller, R., Neilan, B.A., Nett, M., Nielsen, J., O'Gara, F., Oikawa, H.,

489

Osbourn, A., Osburne, M.S., Ostash, B., Payne, S.M., Pernodet, J.L., Petricek, M., Piel, J.,

490

Ploux, O., Raaijmakers, J.M., Salas, J.A., Schmitt, E.K., Scott, B., Seipke, R.F., Shen, B.,

491

Sherman, D.H., Sivonen, K., Smanski, M.J., Sosio, M., Stegmann, E., Süssmuth, R.D.,

492

Tahlan, K., Thomas, C.M., Tang, Y., Truman, A.W., Viaud, M., Walton, J.D., Walsh, C.T.,

493

Weber, T., van Wezel, G.P., Wilkinson, B., Willey, J.M., Wohlleben, W., Wright, G.D.,

494

Ziemert, N., Zhang, C., Zotchev, S.B., Breitling, R., Takano, E., and Glöckner, F.O. (2015)

495

Minimum Information about a Biosynthetic Gene cluster. Nat Chem Biol. 11, 625-631.

496

30. Entfellner, E., Frei, M., Christiansen, G., Deng, L., Blom, J., and Kurmayer, R. (2017)

497

Evolution of anabaenopeptin peptide structural variability in the cyanobacterium

498

Planktothrix. Front Microbiol. 8, 219.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 35

499

31. Fewer, D.P., Jokela, J., Rouhiainen, L., Wahlsten, M., Koskenniemi, K., Stal, L.J., Sivonen,

500

K. (2009) The non-ribosomal assembly and frequent occurrence of the protease inhibitors

501

spumigins in the bloom-forming cyanobacterium Nodularia spumigena. Mol Microbiol. 73,

502

924-937.

503

32. Fujii, K., Sivonen, K., Adachi, K., Noguchi, K., Sano, H., Hirayama, K., Suzuki, M.,

504

Harada, K-I. (1997) Comparative study of toxic and non-toxic cyanobacterial products:

505

novel peptides from toxic Nodularia spumigena AV1. Tetrahedron Lett. 31, 5525–5528.

506

33. Okino, T., Matsuda, H., Murakami, M., Yamaguchi, K. (1993) Microginin, an angiotensin-

507

converting enzyme inhibitor from the blue-green alga Microcystis aeruginosa. Tetrahedron

508

Lett. 34, 501-504.

509

34. Rohrlack, T., Christoffersen, K., Hansen, P.E., Zhang, W., Czarnecki, O., Henning, M.,

510

Fastner, J., Erhard, M., Neilan, B.A., Kaebernick, M. (2003) Isolation, characterization, and

511

quantitative analysis of Microviridin J, a new Microcystis metabolite toxic to Daphnia. J

512

Chem Ecol. 29, 1757-1770.

513

35. Walther, T., Renner, S., Waldmann, H., and Arndt, H.D. (2009) Synthesis and structure-

514

activity correlation of a brunsvicamide-inspired cyclopeptide collection. Chembiochem 10,

515

1153-1162.

516

36. Genuário, D.B., Andreote, A.P.D., Vaz, M.G.M.V., and Fiore, M.F. (2017) Heterocyte-

517

forming cyanobacteria from Brazilian saline-alkaline lakes. Mol Phylogenet Evol. 109, 105–

518

112.

519 520 521 522

37. Kotai, J. (1972) Instructions for preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Research, Oslo, Norway. p 1–5. 38. Seemann, T. (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 20682069.


Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


523

39. Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K.,

524

Gerdes, S., Glass, E.M., Kubal, M., Meyer, F., Olsen, G.J., Olson,f R., Osterman, A.L.,

525

Overbeek, R.A., McNeil, L.K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G.D., Reich,

526

C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A., and Zagnitko, O. (2008) The RAST

527

Server: rapid annotations using subsystems technology. BMC Genomics 9, 75.

528

40. Overbeek, R., Olson, R., Pusch, G.D., Olsen, G.J., Davis, J.J., Disz, T., Edwards, R.A.,

529

Gerdes, S., Parrello, B., Shukla, M., Vonstein, V., Wattam, A.R., Xia, F., and Stevens, R.

530

(2014) The SEED and the Rapid Annotation of microbial genomes using Subsystems

531

Technology (RAST). Nucleic Acids Res. 42, D206-214.

532

41. Brettin, T., Davis, J.J., Disz, T., Edwards, R.A., Gerdes, S., Olsen, G.J., Olson, R.,

533

Overbeek, R., Parrello, B., Pusch, G.D., Shukla, M., Thomason, J.A. 3rd, Stevens, R.,

534

Vonstein, V., Wattam, A.R., and Xia, F. (2015) RASTtk: a modular and extensible

535

implementation of the RAST algorithm for building custom annotation pipelines and

536

annotating batches of genomes. Sci Rep. 5, 8365.

537

42. Medema, M.H., Blin, K., Cimermancic, P., de Jager, V., Zakrzewski, P., Fischbach, M.A.,

538

Weber, T., Breitling, R., and Takano, E. (2011) antiSMASH: Rapid identification,

539

annotation and analysis of secondary metabolite biosynthesis gene clusters. Nucleic Acids

540

Res. 39, W339-346.

541

43. Blin, K., Medema, M.H., Kazempour, D., Fischbach, M.A., Breitling, R., Takano, E., and

542

Weber, T. (2013) antiSMASH 2.0 — a versatile platform for genome mining of secondary

543

metabolite producers. Nucleic Acids Res. 41, W204-212.

544

44. Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H.U., Bruccoleri, R., Lee, S.Y., Fischbach,

545

M.A., Müller, R., Wohlleben, W., Breitling, R., Takano, E., and Medema, M.H. (2015)

546

antiSMASH 3.0 — a comprehensive resource for the genome mining of biosynthetic gene

547

clusters. Nucleic Acids Res. 43, W237-243.



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548 549

Page 24 of 35

45. Rutherford, K., Parkhill, J., Crook, J., Horsnell, T., Rice, P., Rajandream, M.A., and Barrell, B. (2006) Artemis: sequence visualization and annotation. Bioinformatics 16, 944-945.

550

46. Bachmann, B.O., and Ravel, J. (2009) Methods for in silico prediction of microbial

551

secondary metabolic pathways from DNA sequence data. Methods in Enzymology 458, 181-

552

217.

553

47. Rausch, C., Weber, T., Kohlbacher, O., Wohlleben, W., and Huson, D.H. (2005) Specificity

554

prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using

555

transductive support vector machines (TSVMs). Nucleic Acids Res. 33, 5799-5808.

556

48. Röttig, M., Medema, M.H., Blin, K., Weber, T., Rausch, C., and Kohlbacher, O. (2011)

557

NRPSpredictor2—a web server for predicting NRPS adenylation domain specificity.

558

Nucleic Acids Res. 39, W362-W367.

559 560

49. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 30, 2725-2729.

561 562


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563

Tables

564

Table 1. The anabaenopeptins (anabaenopeptin, schizopeptin, and nostamides) and namalides

565

produced by Nostoc sp. CENA543. [M+H]+ is the exact mass and ∆ is difference between exact and

566

measured mass. RI = relative [M+H]+ intensity within each peptide group.

No Peptide

[M+H]+

∆

Subunits

RI

(m/z)

(ppm)

1

2

3

4

5

6

1 Schizopeptin 791 2 Nostamide B

792.46544 806.48109

−2.3 −4.4

Ile Ile

Lys Lys

Ile Ile

Hph Hph

NMeAla NMeAla

Phe Hph

3 Anabaenopeptin 807 4 Nostamide C

808.46035 778.44979

−2.7 −1.5

Ile Ile

Lys Lys

Ile Val

Hty Hph

NMeAla NMeAla

Phe Phe

5 Nostamide D 6 Nostamide E

778.44980 778.44981

−2.7 −3.1

Val Lys Ile Lys

Ile Ile

Hph Hph

NMeAla Ala

Phe Phe

1

Simultaneous Production of Anabaenopeptins and Namalides by the

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