Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

Abstract

Background: Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons.

Results: A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared.

Conclusions: Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.

Fig. 1

Predicted hap biosynthetic gene cluster, including domain organisation and biosynthetic pathway of hapalosin. The hap gene cluster is proposed to encode an initiation module with an AS domain for selection of octanoic acid as the starter unit, two NRPS modules and two PKS modules

Fig. 2

Cyanobactin gene cluster from W. intricata UH strain HT-29-1 and precursor analysis. a The cyanobactin gene cluster from W. intricata UH strain HT-29-1 was aligned with the ten gene cluster Nostoc spongiaeforme var. tenue. b Alignments of cyanobactin precursor peptides encoding multiple cyanobactins with precursor peptide sequence from W. intricata UH strain HT-29-1. The highly conserved LAELSEE motif is indicated above the precursor peptide sequences. The core peptide sequences are indicated in the box. Copies of each core peptide sequence range from one to four copies. W. intricata UH strain HT-29-1 encodes two different cyanobactins, with one (TAACAG) and three (TAACAC) copies of each core peptide. LynE from aestuaramide gene cluster (Lyngbya sp. PCC 8106) [94, 95], McaE from microcyclamide gene cluster (Microcystis aeruginosa PCC 7806, PCC 9809 and PCC 9806 respectively) [72, 96], TruE1,2,3 from trunkamide gene cluster (Prochloron) [94], PatE from patellamide gene cluster (Prochloron) [67], VirE from viridisamide gene cluster (Oscillatoria nigro-viridis PCC 7112) [72], AgeE from aeruginosamide gene cluster (Microcystis aeruginosa PCC 9432) [72], TenE from tenuecyclamide gene cluster (Nostoc spongiaeforme var. tenue) [68]; HT291_05652 from putative cyanobactin biosynthetic gene cluster (Westiella intricata UH strain HT-29-1)

Fig. 3

Microviridin gene cluster from Fischerella sp. PCC 9339 and precursor analysis. a The microviridin gene cluster from Fischerella sp. PCC 9339 was aligned with the mvd gene cluster from Planktothrix agardhii NIVA-CYA 126/8. b Alignment of selected known microviridin precursor peptide sequences with new putative microviridin precursor peptide sequences from Fischerella sp. PCC 9339. The PFFARFL region of the leader peptide and the conserved core peptide region are identified in the boxes. MdnA prepeptide sequences from uncultured Microcystis sp. clones pRus01-07 (GenBank: KF742389-KF742395), uncultured Microcystis sp. clones pFos15 and 19 (GenBank: KF742386 and KF742388), Microcystis UOWOCC MRC (GenBank: CAQ16121), Microcystis aeruginosa NIES-298 (GenBank: CAQ16116), Microcystis aeruginosa NIES-843 (GenBank: BAG02233) and MvdE from Planktothrix agardhii NIVA-CYA 126/8 (GenBank: ACC54551-ACC54552)

Fig. 4

Examples of bacteriocin gene clusters from the Subsection V cyanobacteria. The six different groups (classified according to Wang et al. [34]) identified from the Subsection V cyanobacteria are represented. The group I gene cluster shown was identified from W. intricata UH strain HT-29-1; group II gene cluster shown was identidied from M. repens PCC 10914; the group III and IV gene clusters shown were identidied from H. welwitschii UH strain IC-52-3; the group V gene cluster shown was identified from Fischerella sp. PCC 9431 and the group VI gene cluster shown was identified from Fischerella sp. PCC 9605. Putative precursor genes are represented by a red arrow, HlyD genes are represented by orange arrow, SurA genes are represented by green arrow, ABC transporter genes are represented by green arrow, other modification enzymes are represented by purple arrow, S8 peptidase genes are represented by yellow arrow and LanM genes are represented by pink arrow. Domains involved in cyanobacterial bacteriocin production and modification are highlighted under each gene (domain names derived from the Conserved Domain Database [48]

Fig. 5

MAA and scytonemin biosynthetic gene clusters identified from Subsection V cyanobacteria. a) mys gene cluster from Anabaena variabilis ATCC 29413. b) mys gene cluster identified from Fischerella muscicola SAG 1427-1 and Fischerella sp. PCC 9339. c) mys gene cluster with C domain encoded in NRPS gene identifed from Chlorogloeopsis sp. PCC 9212, Chlorogloeopsis fritschii PCC 6912 and Mastigocoleus testarum BC008. d) mys with additional gene located downstream from NRPS gene identified from Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3 and Fischerella sp. PCC 9431. e) scy gene cluster from Mastigocladopsis repens PCC 10914 (Locus Tag: Mas10914DRAFT_xxxx) aligned with scy gene cluster from Nodularia spumigena CCY9414. Scytonemin core genes are represented by green arrows, regulatory proteins in red, aromatic amino acid biosynthetic genes are blue, other hypothetical genes are represented in yellow, and transposase gene is highlighted in silver arrow. f) Chemical structures of the MAA shinorine and scytonemin

Fig. 6

Additional gene clusters identified from Subsection V cyanobacterial genomes. a Hydrocarbon biosynthetic gene cluster identified from all the Subsection V cyanobacterial genomes. b Geosmin gene cluster identified from W. intricata UH strain HT-29-1, H. welwitschii UH strain IC-52-3, Fischerella sp. PCC 9431 and F. muscicola SAG 1427–1. c Sesquiterpene gene cluster from Fischerella sp. JSC-11, F. thermalis PCC 7521, F. muscicola PCC 7414and Fischerella sp. PCC 9605. d Sesquiterpene gene cluster with hypothetical protein instead of cytochrome p450 identified from Fischerella sp. PCC 9339, W. intricata UH strain HT-29-1, H. welwitschii UH strain IC-52-3 and Fischerella sp. PCC 9431. e Squalene gene cluster encoding phytoene desaturase identified in all Subsection V cyanobacterial genomes. f Squalene gene cluster encoding squalene hopene cyclase identified in all Subsection V cyanobacterial genomes except M. testarum BC008 and M. repens PCC 10914. g Squalene gene cluster encoding squalene hopene and hopene-assocciated glycosyltransferase identified in Chlorogloeopsis sp. PCC 9212, C. fritschii PCC 6912 and M. repens PCC 10914 (squalene synthase gene not clustered in M. repens PCC 10914). h) Unique squalene gene cluster identified from M. testarum BC008 genome. i) Chemical structures of heptadecane, geosmin, 8a-epi-selinene and squalene