Identification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339.

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of l-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

A Plastic Biosynthetic Pathway for the Production of Structurally Distinct Microbial Sunscreens.

Mycosporine-like amino acids (MAAs) are small, colorless, and water-soluble secondary metabolites. They have high molar extinction coefficients and a unique UV radiation absorption mechanism that make them effective sunscreens. Here we report the discovery of two structurally distinct MAAs from the lichen symbiont strain Nostoc sp. UHCC 0926. We identified these MAAs as aplysiapalythine E (C23H38N2O15) and tricore B (C34H53N4O15) using a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy. We obtained a 8.3 Mb complete genome sequence of Nostoc sp. UHCC 0926 to gain insights into the genetic basis for the biosynthesis of these two structural distinct MAAs. We identified MAA biosynthetic genes encoded in three separate locations of the genome. The organization of biosynthetic enzymes in Nostoc sp. UHCC 0926 necessitates a branched biosynthetic pathway to produce two structurally distinct MAAs. We detected the presence of such discontiguous MAA biosynthetic gene clusters in 12% of the publicly available complete cyanobacterial genomes. Bioinformatic analysis of public MAA biosynthetic gene clusters suggests that they are subject to rapid evolutionary processes resulting in highly plastic biosynthetic pathways that are responsible for the chemical diversity in this family of microbial sunscreens.

A cylindrospermopsin-producing cyanobacterium isolated from a microbial mat in the Baltic Sea.

Toxic benthic mats of cyanobacteria are associated with water quality problems and animal poisonings around the world. A strain of the filamentous cyanobacterial genus Kamptonema was isolated from a water bloom in the Baltic Sea four decades ago and later shown to produce cylindrospermopsins. However, the exact habitat of this strain remains unclear and cylindrospermopsins have not yet been reported from water blooms in the Baltic Sea. Here, we report the isolation of Kamptonema sp. UHCC 0994 from a benthic microbial mat collected in shallow water on the coast of Helsinki. We obtained draft genome sequences for the Kamptonema spp. PCC 7926 and UHCC 0994 strains that were isolated from the Baltic Sea. These genomes were 90-96% similar to previously studied Kamptonema sp. PCC 6506 and Kamptonema formosum PCC 6407, which were isolated from benthic and North American freshwater environments, respectively. The genomes of all four Kamptonema strains encode complete cylindrospermopsin biosynthetic gene clusters. We detected the production of cylindrospermopsin and 7-epi-cylindrospermopsin in the four Kamptonema strains using high-resolution liquid chromatography mass spectrometry. The four strains encode genes for producing gas vesicles distributed in two to three different regions of their genomes. Kamptonema spp. UHCC 0994 and PCC 7926 have both retained the ability to regulate their buoyancy when grown in liquid culture. Together this suggests that these toxic cyanobacteria may exhibit a tychoplanktic lifestyle in the Baltic Sea. This study suggests that microbial mats containing cyanobacteria could be a source of environmental toxins in the Baltic Sea.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Biochemical characterization of a cyanobactin arginine-N-prenylase from the autumnalamide biosynthetic pathway.

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

Chemical diversity and cellular effects of antifungal cyclic lipopeptides from cyanobacteria.

Cyanobacteria produce a variety of chemically diverse cyclic lipopeptides with potent antifungal activities. These cyclic lipopeptides have an amphipathic structure comprised of a polar peptide cycle and hydrophobic fatty acid side chain. Many have antibiotic activity against a range of human and plant fungal pathogens. This review article aims to summarize the present knowledge on the chemical diversity and cellular effects of cyanobacterial cyclic lipopeptides that display antifungal activity. Cyclic antifungal lipopeptides from cyanobacteria commonly fall into four structural classes; hassallidins, puwainaphycins, laxaphycins, and anabaenolysins. Many of these antifungal cyclic lipopeptides act through cholesterol and ergosterol-dependent disruption of membranes. In many cases, the cyclic lipopeptides also exert cytotoxicity in human cells, and a more extensive examination of their biological activity and structure-activity relationship is warranted. The hassallidin, puwainaphycin, laxaphycin, and anabaenolysin structural classes are unified through shared complex biosynthetic pathways that encode a variety of unusual lipoinitiation mechanisms and branched biosynthesis that promote their chemical diversity. However, the biosynthetic origins of some cyanobacterial cyclic lipopeptides and the mechanisms, which drive their structural diversification in general, remain poorly understood. The strong functional convergence of differently organized chemical structures suggests that the production of lipopeptide confers benefits for their producer. Whether these benefits originate from their antifungal activity or some other physiological function remains to be answered in the future. However, it is clear that cyanobacteria encode a wealth of new cyclic lipopeptides with novel biotechnological and therapeutic applications.

The structure and biosynthesis of heinamides A1-A3 and B1-B5, antifungal members of the laxaphycin lipopeptide family.

Laxaphycins are a family of cyclic lipopeptides with synergistic antifungal and antiproliferative activities. They are produced by multiple cyanobacterial genera and comprise two sets of structurally unrelated 11- and 12-residue macrocyclic lipopeptides. Here, we report the discovery of new antifungal laxaphycins from Nostoc sp. UHCC 0702, which we name heinamides, through antimicrobial bioactivity screening. We characterized the chemical structures of eight heinamide structural variants A1-A3 and B1-B5. These variants contain the rare non-proteinogenic amino acids 3-hydroxy-4-methylproline, 4-hydroxyproline, 3-hydroxy-d-leucine, dehydrobutyrine, 5-hydroxyl ß-amino octanoic acid, and O-carbamoyl-homoserine. We obtained an 8.6-Mb complete genome sequence from Nostoc sp. UHCC 0702 and identified the 93 kb heinamide biosynthetic gene cluster. The structurally distinct heinamides A1-A3 and B1-B5 variants are synthesized using an unusual branching biosynthetic pathway. The heinamide biosynthetic pathway also encodes several enzymes that supply non-proteinogenic amino acids to the heinamide synthetase. Through heterologous expression, we showed that (2S,4R)-4-hydroxy-l-proline is supplied through the action of a novel enzyme LxaN, which hydroxylates l-proline. 11- and 12-residue heinamides have the characteristic synergistic activity of laxaphycins against Aspergillus flavus FBCC 2467. Structural and genetic information of heinamides may prove useful in future discovery of natural products and drug development.

CyanoMetDB, a comprehensive public database of secondary metabolites from cyanobacteria.

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.

Shared PKS Module in Biosynthesis of Synergistic Laxaphycins.

Cyanobacteria produce a wide range of lipopeptides that exhibit potent membrane-disrupting activities. Laxaphycins consist of two families of structurally distinct macrocyclic lipopeptides that act in a synergistic manner to produce antifungal and antiproliferative activities. Laxaphycins are produced by range of cyanobacteria but their biosynthetic origins remain unclear. Here, we identified the biosynthetic pathways responsible for the biosynthesis of the laxaphycins produced by Scytonema hofmannii PCC 7110. We show that these laxaphycins, called scytocyclamides, are produced by this cyanobacterium and are encoded in a single biosynthetic gene cluster with shared polyketide synthase enzymes initiating two distinct non-ribosomal peptide synthetase pathways. The unusual mechanism of shared enzymes synthesizing two distinct types of products may aid future research in identifying and expressing natural product biosynthetic pathways and in expanding the known biosynthetic logic of this important family of natural products.

Dereplication of Natural Products with Antimicrobial and Anticancer Activity from Brazilian Cyanobacteria.

Cyanobacteria are photosynthetic organisms that produce a large diversity of natural products with interesting bioactivities for biotechnological and pharmaceutical applications. Cyanobacterial extracts exhibit toxicity towards other microorganisms and cancer cells and, therefore, represent a source of potentially novel natural products for drug discovery. We tested 62 cyanobacterial strains isolated from various Brazilian biomes for antileukemic and antimicrobial activities. Extracts from 39 strains induced selective apoptosis in acute myeloid leukemia (AML) cancer cell lines. Five of these extracts also exhibited antifungal and antibacterial activities. Chemical and dereplication analyses revealed the production of nine known natural products. Natural products possibly responsible for the observed bioactivities and five unknown, chemically related chlorinated compounds present only in Brazilian cyanobacteria were illustrated in a molecular network. Our results provide new information on the vast biosynthetic potential of cyanobacteria isolated from Brazilian environments.

Biosynthesis of the Bis-Prenylated Alkaloids Muscoride A and B.

Prenylation is a common step in the biosynthesis of many natural products and plays an important role in increasing their structural diversity and enhancing biological activity. Muscoride A is a linear peptide alkaloid that contain two contiguous oxazoles and unusual prenyl groups that protect the amino- and carboxy-termini. Here we identified the 12.7 kb muscoride (mus) biosynthetic gene clusters from Nostoc spp. PCC 7906 and UHCC 0398. The mus biosynthetic gene clusters encode enzymes for the heterocyclization, oxidation, and prenylation of the MusE precursor protein. The mus biosynthetic gene clusters encode two copies of the cyanobactin prenyltransferase, MusF1 and MusF2. The predicted tetrapeptide substrate of MusF1 and MusF2 was synthesized through a novel tandem cyclization route in only eight steps. Biochemical assays demonstrated that MusF1 acts on the carboxy-terminus while MusF2 acts on the amino-terminus of the tetrapeptide substrate. We show that the MusF2 enzyme catalyzes the reverse or forward prenylation of amino-termini from Nostoc spp. PCC 7906 and UHCC 0398, respectively. This finding expands the regiospecific chemical functionality of cyanobactin prenyltransferases and the chemical diversity of the cyanobactin family of natural products to include bis-prenylated polyoxazole linear peptides.

A Report on Finding a New Peptide Aldehyde from Cyanobacterium Nostoc sp. Bahar M by LC-MS and Marfey's Analysis.

BACKGROUND: Cyanobacteria have a worldwide distribution in the terrestrial habitats, occurring predominantly on the surface of the soils, stones, rocks, and trees, practically in moist, neutral or alkaline aeries. The unique natural and bioactive compounds from cyanobacteria with various biological activities and an extensive range of chemical classes have a significant capability for expansion of the pharmaceuticals and other biomedical purposes. OBJECTIVES: Regardless of the progresses in our knowledge on cyanobacteria, however, cyanobacteria are still viewed as an unexplored source of potential drugs. In this study presence of bioactive compounds among the cyanobacteria culture collection of Iran, where a wide variety of strains can be found, was investigated. MATERIAL AND METHODS: We explored one Nostoc strain isolated from rice fields in Golestan province of northern Iran for searching for novel products. The chemical construction of the new bioactive compound was clarified by application of liquid chromatography-mass spectrometer (LC-MS) and Marfey's analysis of the degradation products. RESULTS: We found a novel peptide aldehyde compound from a hydrophilic extract of the Nostoc sp. Bahar_M, which is composed of the three subunits, 2-hydroxy-4-(4-hydroxyphenyl) butanoic acid (Hhpba), L-Ile, and L-argininal. According to the structural information, we predicted that the novel peptide-aldehyde compound probably to be trypsin inhibitors. CONCLUSIONS: Results demonstrated that terrestrial cyanobacteria are a promissing resource of bioactive natural products.

Characterization of the interaction of the antifungal and cytotoxic cyclic glycolipopeptide hassallidin with sterol-containing lipid membranes.

Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causing a lytic/necrotic cell death with rapid presence of disintegrated outer membrane, accompanied by internalization of small molecules such as propidium iodide into the cells. Furthermore, artificial liposomal membrane assay showed that hassallidin D selectively targets sterol-containing membranes. Finally, in silico membrane modeling allowed us to study the interaction between hassallidin D and membranes in detail, and confirm the role of cholesterol for hassallidin-insertion into the membrane. This study demonstrates the mechanism of action of the natural compound hassallidin, and gives further insight into how bioactive lipopeptide metabolites selectively target eukaryotic cell membranes.

The Biosynthesis of Rare Homo-Amino Acid Containing Variants of Microcystin by a Benthic Cyanobacterium.

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic strain Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.

Identification and structural analysis of cereal arabinoxylan-derived oligosaccharides by negative ionization HILIC-MS/MS.

Recent works provide evidence of the prebiotic potential of arabinoxylan-derived oligosaccharides (A)XOS. In this study, we developed a structural analysis for cereal-derived (A)XOS by negative ionization HILIC-MS/MS. Initially, we assessed twelve (A)XOS samples of known structures with different linkage positions and branching points by direct-infusion negative ESI-MSn. We subsequently developed the negative ion HILIC-MS/MS with a post-column addition of ammonium chloride. The selected (A)XOS represented both linear (arabinofuranosyl residue linked to the non-reducing end of xylooligosaccharide) and branched structures. Each (A)XOS sample produced a specific spectrum in negative ion ESI-MSn. By analyzing cross-ring fragment ions, we determined the linkage positions of linear (A)XOS. The presence or absence of diagnostic ions in the MS3 allowed us to detect different branches (O-2- or/and O-3-linked arabinofuranosyl with/or without O-4-linked xylopyranosyl at the non-reducing end). Furthermore, we could identify all analyzed samples by HILIC-MS/MS, based on the formed spectral library and chromatographic retention times.

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides.

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

N-Prenylation of Tryptophan by an Aromatic Prenyltransferase from the Cyanobactin Biosynthetic Pathway.

Aromatic prenylation is an important step in the biosynthesis of many natural products and leads to an astonishing diversity of chemical structures. Cyanobactin pathways frequently encode aromatic prenyltransferases that catalyze the prenylation of these macrocyclic and linear peptides. Here we characterized the anacyclamide ( acy) biosynthetic gene cluster from Anabaena sp. UHCC-0232. Partial reconstitution of the anacyclamide pathway, heterologous expression, and in vitro biochemical characterization demonstrate that the AcyF enzyme, encoded in the acy biosynthetic gene cluster, is a Trp N-prenyltransferase. Bioinformatic analysis suggests the monophyletic origin and rapid diversification of cyanobactin prenyltransferase enzymes and the multiple origins of N-1 Trp prenylation in prenylated natural products. The AcyF enzyme displayed high flexibility toward a range of Trp-containing substrates and represents an interesting new tool for biocatalytic applications.

Sphaerocyclamide, a prenylated cyanobactin from the cyanobacterium Sphaerospermopsis sp. LEGE 00249.

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, sphaerocyclamide (1), from Sphaerospermopsis sp. LEGE 00249. The sphaerocyclamide biosynthetic gene cluster (sph) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the sph gene cluster in Escherichia coli confirmed the connection between genomic and mass spectrometric data. Unambiguous establishment of the orientation and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis.

Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules.