Multicomponent Microscale Biosynthesis of Unnatural Cyanobacterial Indole Alkaloids

Genome
sequencing and bioinformatics tools have facilitated the
identification and expression of an increasing number of cryptic biosynthetic
gene clusters (BGCs). However, functional analysis of all components
of a metabolic pathway to precisely determine biocatalytic properties
remains time-consuming and labor intensive. One way to speed this
process involves microscale cell-free protein synthesis (CFPS) for
direct gene to biochemical function analysis, which has rarely been
applied to study multicomponent enzymatic systems in specialized metabolism.
We sought to establish an in vitro transcription/translation
(TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type
natural products (cNPs) because of their diverse bioactivity profiles
and complex structural diversity. Using a CFPS system including a
plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the
central prenylated intermediate (3GC) in the presence of exogenous
geranyl-pyrophosphate (GPP) and cis-indole isonitrile.
Further addition of a plasmid bearing the famC1 Stig
cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which
was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases
(FamC1–C4) produced hapalindole U and hapalindole H, while
FisC identified from Fischerella sp. SAG46.79 generated
12-epi-fischerindole U. The CFPS system was further
employed to screen six unnatural halogenated cis-indole
isonitrile substrates using FamC1 and FisC, and the reactions were
scaled-up using chemoenzymatic synthesis and identified as 5- and
6-fluoro-12-epi-hapalindole U, and 5- and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents
an effective, high throughput strategy to determine the functional
role of biosynthetic enzymes from diverse natural product BGCs.