## 5 Biosynthesis of Indole Alkaloids from the Stigonematales
The 42 kbp _amb_ gene cluster, encoding 32 proteins, was confirmed by both bioinformatics, and _in vitro_ enzyme studies, to be involved in the biosynthesis of ambiguines including steps common to all hapalindole alkaloids, and specifically genes associated with biosynthesis of tryptophan, prenylation precursors (_i.e._, isopentyl pyrophosphate and dimethylallyl pyrophosphate [DMAPP]) and the indole-isonitrile intermediate, as well as key steps in the prenylation of tetracyclic hapalindoles to ambiguines ([PERSON] _et al._, 2013). Furthermore, the presence of several oxygenases suggests a contribution to the \"late stage\" biosynthetic steps characteristic of the diversity of ambiguines. Concurrently, characterization of the _wel_ cluster from _H. wellwitschi_[PERSON] _et al._ (2014) identified genes related to the biosynthesis of the wellwitindolinones including key precursors, namely of 3-(Z-2'-isocyanoether))indole, and the putative monoterenoid building blocks. Gene products associated with indole-isonitrile biosynthesis (_i.e._, AmbI1-3 and Well1-3) in both _amb_ and _wel_ gene clusters were evaluate in enzyme studies, and specifically found to produce only the _cis_ form of the indole-isonitrile intermediate (Figure 13). Finally in addition to these common genes, the _wel_ gene cluster included apparent methyltransferases suggested to have a role in the \(N\)-methylation of several of the wellwitindolinones, and were demonstrated in _in vitro_ enzyme studies to, in fact, function as such. Most generally, both studies support condensation of geranyl pyrophosphate (GPP) and the indole-isonitrile, rather than conversion of GPP to \(\beta\)-ocimene, as previously proposed ([PERSON] _et al._, 2013; [PERSON] _et al._, 2013). A particularly comprehensive, subsequent study ([PERSON] _et al._, 2013) coupled whole genome sequencing of multiple stigonematalean species--including _Fischerella, Hapalosiphon_ and _Westiella_--and previously published, and unpublished, sequence data for gene clusters associated with hapalindole-type alkaloid biosynthesis (e.g., _amb_, _wel_[PERSON] _et al._, 2014) to elaborate a unified biosynthetic pathway. Comparative studies,accordingly, identified 19 gene sets, with 92% sequence identity at the protein level, which were common to all gene clusters (and species/strains) investigated. Supporting the proposed early steps of the biosynthesis of the alkaloids, these \"core\" genes include those involved in tryptophan biosynthesis, and isonitrile biosynthesis genes involved in the formation of the 3-(\(Z\)-\(2^{\prime}\)-isocyanoethenyl)indole precursor. Notably in this study, the gene products of the latter, and specifically Well1/3, were found to produce _in vitro_ both _cis_ and _trans_ isomers of the indole-isonitrile, and were thereby suggested to explain the stereodisposition between C10 and C11 observed among the haplindole-type alkaloids. However, concurrent studies suggested (as discussed above) that only the _cis_ form (_i.e.,_ Z-isomer) is produced, and subsequently utilized, in the condensation [49; 55]. This discrepancy was attributed to differences in incubation time of feeding experiments in the two studies. However, it should be noted that Well genes assessed in the conflicting studies [49; 57] were, in fact, isolated from two different genera/species (_i.e., H. welwitschii_ and _W. intricate_), and differ to some extent in their sequence; and it is possible, therefore, that differences in enzymatic activity could also reflect phylogenetic differences in the biosynthesis. Conserved genes additionally included those involved in isoprene biosynthesis, specifically including geranyl phosphate synthases, as well as aromatic prenyltransferase which was proposed to be involved in the condensation of the indole-isonitrile to GPP (Figure 15) although this could not be confirmed in the study.