BackgroundThe hapalindole-type family of natural products is a group of hybrid isoprenoid-indole alkaloids, produced solely by members of the Subsection V cyanobacterial strains. This family broadly includes the hapalindoles, welwitindolinones, fisherindoles and ambiguines amongst others, all of which have an isonitrile- or isothiocyanate-containing indole alkaloid skeleton, with a cyclized isoprene unit. The hapalindoles are diversified into the welwitindolinones, fischerindoles and ambiguines through the employment of tailoring oxygenase, methyltransferase and prenyltransferase enzymes. We compare the genetic basis for the biosynthesis of this diverse group of natural products and identify key early biosynthetic intermediates.ResultsWhole genome sequencing of freshwater and terrestrial cyanobacteria Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp. ATCC 43239 led to the identification of a candidate hapalindole-type gene cluster in each strain. These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes. We present detailed comparative bioinformatic analysis of the gene clusters and the biosynthesis of a pivotal indole-isonitrile intermediate resulting in both cis and trans geometrical isomers. Enzyme analyses and metabolite extractions from two hapalindole-producing Fischerella strains indicate the presence of cis and trans indole-isonitriles as biosynthetic intermediates in the early steps of the pathway.ConclusionsInterestingly, the organization of the welwitindolinone gene cluster is conserved in all producing strains but distinct from the hapalindole and ambiguine clusters. Enzymatic assays using WelI1 and WelI3 from Westiella intricata UH strain HT-29-1 demonstrated the ability to catalyze the formation of both cis and trans geometrical isomers when using a cell lysate. The enzymatic and metabolic characterization of both cis and trans indole-isonitrile intermediates implies conservation of their stereochemical integrity towards members of the ambiguine and welwitindolinone products. In summary, we present data that supports a unified biosynthetic pathway towards hapalindoles in nine individual species of cyanobacteria. Diversification of the pathway occurs later through the employment of specialized enzymatic steps towards fischerindoles, ambiguines and welwitindolinones.

Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-l-Trp-l-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1'-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.

Bacteria can utilize a mammalian host siderophore to usurp host iron; however, the host can respond by down-regulating siderophore expression and up-regulating expression of an inhibitory siderophore-binding protein.