Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids

Abstract

The formation of C-C bonds in an enantioselective fashion to create complex polycyclic scaffolds in the hapalindole- and fischerindole- type alkaloids from Stigonematales cyanobacteria represents a compelling and urgent challenge in adapting microbial biosynthesis as a catalytic platform in drug development. Here we determine the biochemical basis for tri- and tetracyclic core formation in these secondary metabolites, involving a new class of cyclases that catalyze a complex cyclization cascade.

Figure 1. Select hapalindole -type alkaloids

a) Biosynthetic pathway leading to 12-epi- hapalindole U; b) representative indole alkaloids from related cyanobacterial metabolic pathways illustrating the variant polycyclic core systems.^–

Figure 2. Gene clusters identified from hapalindole producing strains

fam gene cluster from ambiguine producing strain Fischerella ambigua UTEX 1903; fil gene cluster from ambiguine producing strain Fischerella sp. IL-199-3-1 (this study); hpi gene cluster from hapalindole producing strain Fischerella sp. ATCC 43239 (this nomenclature has been revised for consistency with other newly annotated gene clusters); fim gene cluster from fischerindole producing strain Fischerella muscicola UTEX 1829 (this study); fis gene cluster from fischerindole producing strain Fischerella sp. SAG 46.79 (this study). Full annotation of these gene clusters is in Supplementary Tables 2–5.

Figure 3. In vitro assay involving recombinant cyclases to form hapalindole and fischerindole natural products

Assays were conducted in a one-pot enzymatic tandem reaction by incubating substrates indole isonitrile (1) and GPP (2) with FamD2 and the selected cyclases. a) Proteins producing 12-epi-hapalindole U (4); b) protein producing 12-epi-fischerindole U (9) and 12-epi-hapalindole C (7); c) proteins producing hapalindole H (5); cell-free lysate is from UTEX 1903 strain; d) summary of enzymatic hapalindole/fischerindole production determined in this study (see Supplementary Fig. 3 for the structure of 10).