## 5 Biosynthesis of Indole Alkaloids from the Stigonematales
In addition to these common genes, several others seemingly specific to the different sub-types were identified including various putative oxygenases, an aromatic prenylase involved in the C-2 prenylation of the ambiguities (from the _amb_ cluster), and methyltransferases involved in the \(N\)-methylation of the welwitindolinones (from the _vel_ cluster). Finally, a phylogenetic analysis of the strains and the gene clusters--based on both 16s rDNA and relevant, conserved biosynthetic genes (_i.e.,_ prenyltransferase) --indicated a monophyletic group, and thus vertical inheritance, of these biosynthetic genes. This latter finding, thereby, further supports the unique distribution of the indole alkaloids within the Stigonematales. Two very recent studies, based on genomic data, and _in vitro_ enzymatic studies, have attempted to elucidate a potential mechanism for condensation of GPP and the indole-isonitrile precursor [55; 58]. In one study, a homologous series of strictly magnesium-dependent aromatic prenyltransferases (AmbPI, WelP1 and FidP1) was identified across related biosynthetic gene clusters (_i.e., wel_, _amb_ and unpublished _fid_), and specifically shown to direct the formation of 3-geranyl 3-isocyanovinyl indolenine--via condensation of GPP and the \(Z\)-isomer of the indole isonitrile--as a common intermediate [55]. Following an unusual Cope rearrangement, it was suggested that a tricyclic haplindole could be formed via aza-Prins-type cyclization, and subsequently provide a starting point for a wide range of haplindole-type alkaloids (Figure 15). Concurrent studies, however, by [PERSON] _et al._[58] identified the _fam_ gene cluster from _F. ambigua_ (involved in the biosynthesis of ambiguities), and demonstrated through _in vitro_ studies with cell-free lysates, the role of two prenyltransferases (FamD1 and FamD2) and a cyclase (FamC1) within this cluster. It was proposed that FamD2 and FamC1 \"act in concert\" to catalyze the condensation of the indole-isonitrile and GPP via similar Cope rearrangement, and a similar quaternary intermediate, but distinct from the studies by [PERSON] _et al._[55] suggested the formation of a tetracyclic haplindole core, and namely 12-_epi_-hapalindole U. Subsequent to this, FamD1 was shown to prenylate (with DMAPP) at the C-2 position to give rise to the ambiguines. Furthermore, it was suggested that the cyclase step (via FamC1, or perhaps homologs) could be involved in determining stereochemistry (_i.e.,_ C-12 epimers, C10/C15), and also directing biosynthesis toward the fischerindoles. These findings, in general, reinforce a biosynthesis that circumvents the previously proposed \(\beta\)-ocimene precursor in this key step. Intriguingly, both studies point to dependence of the condensation step on pH and/or Mg\({}^{2+}\), and might, thereby, underscore potential plasticity, relevant to structural diversification, at this step. Finally, recent work from the [PERSON] group [18] has identified a non-heme, iron-dependent halogenase (_i.e._, _welO5_) from the _wel_ gene cluster, and has specifically demonstrated the ability of this enzyme to catalyze chlorination of the C-13 of a tricyclic haplaindole (_i.e._, 12-_epi_-hapalindole C) and fischerindole (_i.e._, 12-_epi_-fischerindole U). This finding not only explains the presence of chlorinated and non-chlorinated congeners found among many of the haplaindole-type alkaloids, but also suggests a role of this chlorination as a first committed step in the subsequent formation of welwitindolinones [18] (Figure 14), and further explains, therefore, the previously unexplained absence of dichloro variants within this sub-class.