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.Peer reviewe

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies

Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis

Peer reviewe

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 beta-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.Peer reviewe

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

<p>Abstract</p> <p>Background</p> <p>Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of <it>mcyB (mcyB1) </it>and <it>mcyC </it>in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus <it>Microcystis</it>.</p> <p>Results</p> <p>Here we undertook a phylogenetic study to investigate the order and timing of recombination between the <it>mcyB1 </it>and <it>mcyC </it>genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the <it>mcyB1 </it>and <it>mcyC </it>adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between <it>mcyB1 </it>and <it>mcyC </it>in strains of the genera <it>Anabaena</it>, <it>Microcystis</it>, and <it>Hapalosiphon</it>. We also find clear evidence for independent adenylation domain conversion of <it>mcyB1 </it>by unrelated peptide synthetase modules in strains of the genera <it>Nostoc </it>and <it>Microcystis</it>. The recombination events replace only the adenylation domain in each case and the condensation domains of <it>mcyB1 </it>and <it>mcyC </it>are not transferred together with the adenylation domain. Our findings demonstrate that the <it>mcyB1 </it>and <it>mcyC </it>adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.</p> <p>Conclusion</p> <p>Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.</p

Comparative Genomics of the Baltic Sea Toxic Cyanobacteria Nodularia spumigena UHCC 0039 and Its Response to Varying Salinity

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spurn/germ UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.Peer reviewe

Description of Aliinostoc alkaliphilum sp. nov. (Nostocales, Cyanobacteria), a New Bioactive Metabolite-Producing Strain from Salina Verde (Pantanal, Brazil) and Taxonomic Distribution of Bioactive Metabolites in Nostoc and Nostoc-like Genera

Cyanobacteria are a group of oxygenic photosynthetic prokaryotes found in almost all habitats on earth including those characterized as extreme environments. It has been observed that the number of studies dealing with the biodiversity of extremophilic cyanobacteria is limited while studies exploring their bioactive potential are even scarcer. The taxonomy of three Nostoc-like cyanobacterial strains isolated from a shallow lake in Brazil was studied by applying a polyphasic approach. The bioactive potential of the strains was also evaluated using antimicrobial susceptibility testing. The metabolites present in the bioactive HPLC fractions were identified by UPLC/ESI/Q-TOF. Based on our phylogenetic inferences in combination with morphological and ecological information, we describe Aliinostoc alkaliphilum sp. nov., exhibiting antibacterial and antifungal activities. The main bioactive metabolite in all three strains was nocuolin A, which represents the first report of this metabolite in Aliinostoc. Our phylogenetic studies also revealed that many bioactive metabolite-producting strains that are currently assigned to Nostoc belong to other distinct evolutionary lineages. These findings highlight the importance of polyphasic approach studies in both cyanobacterial taxonomy and natural product discovery programs