Multiple hapalindoles, GSK-3 inhibitor fischerindoles and welwitindolinones have been reported to be produced by Hapalosiphon welwitschii UH strain IC-52-3, whilst three welwitindolinones have been reported from Westiella intricata UH strain HT-29-1 [10] (Figure 1). We aimed to identify a gene cluster responsible for the biosynthesis of these compounds in each strain, while also screening publicly available cyanobacterial genomes for the presence of the hapalindole-type biosynthetic gene cluster. The genetic analyses were complemented by in vitro enzymatic assays for the isonitrile biosynthesis enzymes WelI1 and WelI3, resulting in the formation of both cis and trans isoforms of

3-(2-isocyanovinyl)indole (hereafter known as indole-isonitrile). Furthermore, the enzymology is supported through structural verification of both cis and trans isoforms of the indole-isonitrile extracted directly from Fischerella sp. and Fischerella ambigua cultures. Results and discussion Whole

genome sequencing of Fischerella sp. ATCC 43239 (hereafter known as FS ATCC43239), Fischerella ambigua UTEX 1903 (hereafter known as FA UTEX1903), Hapalosiphon welwitschii UH strain IC-52-3 (hereafter known as HW IC-52-3) and Westiella intricata UH strain HT-29-1 (hereafter known as WI HT-29-1) was used to identify a gene cluster encoding the biosynthesis of the hapalindoles (precursor molecules for fischerindole, ambiguine and welwitindolinone biosynthesis) in each strain. Dolichyl-phosphate-mannose-protein mannosyltransferase Candidate Tubastatin A manufacturer gene clusters were identified in all four sequenced genomes, and PCR reactions were used to seal any gaps. The wel (welwitindolinone) gene cluster was identified in the genome of WI HT-29-1 (Additional file 1: Table S1), and in the genome of HW IC-52-3 (Additional file

1: Table S2). The hpi (hapalindole) gene cluster was identified in the FS ATCC43239 genome (Additional file 1: Table S3). The ambiguine (amb) gene cluster was recently published by Hillwig et al. [7]. We independently sequenced and identified the amb gene cluster from the genome of FA UTEX1903 as part of this study. While the majority of the nucleotide sequence is 100% identical, some differences upstream of the 3’ end of ambE3 were identified. The amb gene cluster from Hillwig et al. [7] encodes ParA and ParB family chromosome partitioning proteins and transposases, however, the amb gene cluster sequenced in this study does not contain these genes, instead, genes encoding monooxygenases and oxidoreductases were identified (Additional file 1: Table S4). There are currently 11 Subsection V cyanobacteria draft genomes that are publicly available. We screened all Subsection V genomes in an attempt to identify any additional gene clusters encoding the biosynthesis of the hapalindole group of compounds. There has been no reported investigation of hapalindole-type natural products from these strains.