Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria

Abstract

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 These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes.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.

Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis

“…23 We present evidence that points to early stage assembly of cyclo-Trp-Trp DKP as an intermediate that undergoes a regio-and stereoselective C3methyltransfer step resulting in the formation of a subsequent intermediate that is C3′-normal prenylated and N1′-methylated by respective enzymes encoded in the noz pathway with reasonable promiscuity in the order of their occurrence. 23 We present evidence that points to early stage assembly of cyclo-Trp-Trp DKP as an intermediate that undergoes a regio-and stereoselective C3methyltransfer step resulting in the formation of a subsequent intermediate that is C3′-normal prenylated and N1′-methylated by respective enzymes encoded in the noz pathway with reasonable promiscuity in the order of their occurrence.…”

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

“…Although the isonitrile functionality has been found in quite a few natural products, only one biosynthetic pathway has been identified in which one carbon is transferred to an amino group catalyzed by an isonitrile synthase such as IsnA (27)(28)(29)(30). Our proposed pathway for isonitrile synthesis is mechanistically distinct from the known pathway by using a different set of enzymes.…”

“…This isonitrile intermediate is then condensed to both amino groups of Lys promoted by an NRPS and reductively released to form a terminal alcohol product. The identified isonitrile biosynthetic pathway is distinct from the canonical mechanism in which one carbon is transferred to an amino group to form isonitrile catalyzed by an isonitrile synthase (27)(28)(29)(30).…”

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria

“…A related cluster could be assigned to the biosynthesis of welwitindolinone in Hapalosiphon welwitschii [53]. Structural diversity within the hapalindole family is generated by different types of terpene cyclizations, chlorination, methylation, and oxygenation, and further prenylation as suggested by genome sequencing efforts and bioinformatic comparison of stigonematalean strains producing diverse types of hapalindoles [54]. The biochemical basis for this diversity is now being unraveled, and the tailoring enzyme WelO5 of the welwitindolinone pathway could be characterized as the prototype of a novel iron-depending halogenase that acts on free-standing substrates [55].…”