## 5 Biosynthesis of Indole Alkaloids from the Stigonematales  
In support of the impressive chemical diversity observed, a continuing body of knowledge has evolved with respect to the biosynthesis of the haplindole-type alkaloids. Species (and strains within species) of the Stigonematales frequently produce several of the indole alkaloid sub-classes, and the observed isolation of diverse congeners from a single source leads to the conclusion that a similar biosynthetic pathway occurs for these compounds. Indeed, a unified biosynthesis of the haplindole-type alkaloids was accordingly proposed soon after their discovery [45]. However, very recent genomic data have led, in many cases, to considerable revision of this proposed biogenic pathway. In the first assessments of the biosynthesis of the haplindole alkaloids, [PERSON] _et al._[45] specifically suggested that initial steps occurred (Figure 12) by way of a chloronium or hydrogen ion-induced condensation of 3-(\(Z\)-\(2^{\prime}\)-isocyanoetherly)indole (_i.e._, \"indole-isonitrile\") and (\(Z\))-3,7-dimethyl-1,3,6-octatriene (_i.e._, \(\beta\)-ocimene), whereby these precursors are formed, respectively, from tryptophan and geranyl pyrophosphate (GPP), leading to the formation of a presumptive tricyclic haplaindole. Based on this biosynthetic scheme, 12-epimers of the haplanlodes were proposed to be alternatively derived from either \(E\)- or \(Z\)-isomers of \(\beta\)-ocimene (see [49]). Very recent studies, incorporating genomic and other molecular approaches, however, clearly suggest an alternative biosynthetic route, and specifically direct prenylation of the indole-isonitrile by GPP, rather than involvement of \(\beta\)-ocimene (as discussed below). Similarly, the presence of a _cis_ and _trans_ isomers (_i.e._, \(Z\)- and \(E\)-isomers, respectively) of the indole-isonitrile were proposed to underlie the stereochemistry at C-10/11 in haplindoles; however, very recent genomic studies suggest that only the \(Z\)-isomer is a substrate for the condensation step (also discussed below). With respect to the biosynthesis of the indole-isonitrile, it was proposed in early studies that the isonitrile functional group found in this precursor, and consequently, the majority of the sub-classes, is either derived from inorganic cyanide, or cyanide produced from C-2/N-2 of amino acids [50]--as reported, in fact, for cyanobacteria [51]--by way of the tetrahydrorofolate (THF) C1 pathway, and specifically a 5-formimino-THF intermediate. Contribution of amino acid-derived C and N to the isonitrile is supported by studies of haplindole A, and specifically feeding studies of _H. fontinalis_ utilizing isotopically (_i.e._, \({}^{13}\)C, \({}^{15}\)N) labeled glycine [52]. The incorporation of inorganic cyanide is indirectly supported by feeding studies of sponge-derived, isonitrile-containing compounds [53], as well as radiolabeling (_i.e._, \({}^{14}\)C-labeled cyanide) studies in the cyanobacteria. However, attempts to replicate the latter feeding studies with inorganic cyanide labeled with stable isotope (_i.e._, \({}^{13}\)C) were confounded, as the requisite concentrations proved toxic to the cyanobacterial cultures [52]. Moreover,  
Figure 11: Previously described Type B wellwitindolinones (Group 9) from Stigonematales. recent genomic studies [54] suggest an alternative biogenic route for the indole-isonitrile. Identification of a gene cluster (_amb_) associated with ambiguine biosynthesis revealed three homologs (_i.e._, AmblI-3) of previously characterized, bacterial isonitrile synthase genes, and following purification of the gene products--and _in vitro_ enzyme studies--demonstrated specific production of the _cis_-isomer (and, notably, not the _trans_ form) of the indole-isonitrile from tryptophan and ribulose-5-phosphate (as a carbon source) as shown in Figure 13[54]. Similar results were found for homologs from the _vel_ gene cluster associated with biosynthesis of wellwitindolinones [49].