A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Abstract

The next frontier in drug discovery could be the semi-synthesis of non-natural, xenobiotic compounds combining both natural product biosynthesis and synthetic chemistry. However, the required tools and underlying engineering principles are yet to be fully understood. One way to investigate non-natural product biosynthesis is to probe the substrate promiscuity of a clinically relevant biosynthesis pathway. Violacein is a bisindole compound produced by the VioABCDE biosynthesis pathway using L-tryptophan as the starting substrate. Previous studies have shown that violacein exhibits antimicrobial properties, and synthetic analogues of violacein might give rise to new targets for therapeutic development to combat antimicrobial resistance. By adding seven types of tryptophan analogues available commercially, 62 new violacein or deoxyviolacein analogues were generated with a synthetic violacein biosynthesis pathway expressed in Escherichia coli, demonstrating the promiscuity of violacein biosynthesis enzymes. Growth inhibition assays against Bacillus subtilis, a Gram-positive bacterium, were carried out to measure growth inhibitory activity of violacein analogues compared to violacein. In addition, we show that four new 7-chloro analogues of violacein or deoxyviolacein can be generated in vivo by combining the rebeccamycin and violacein biosynthesis pathways and purified 7-chloro violacein was found to have similar growth inhibitory activity compared to violacein. Structural studies of VioA revealed active site residues that are important for catalytic activity, and further pathway recombination with VioA homologues in related bisindole pathways may lead to more efficient enzymes that would accept tryptophan analogues more readily.

A biosynthetic platform for antimalarial drug discovery

“…We have confirmed the viability of the system, ensuring there is no background antiparasitic activity in bacterial solvent extracts lacking violacein. We then tested the biosynthetic violacein extract from E. coli and confirmed its half maximal inhibitory concentration (IC50) in agreement with a commercial violacein standard and previous studies 14 . Finally, as well as using this approach to explore the mode of action of violacein, we show that extracts representing a diverse series of biosynthetically derived variants show varying effects on parasite growth, with 16 of the 28 compound mixtures inhibiting growth to a greater level than the parent violacein molecule.…”

“…S4) 21 . The bacterial extracts were produced by feeding corresponding tryptophan substrates as described previously 14 and violacein concentrations in the extracts were calibrated against a violacein standard. Asexual growth assays were again carried out, testing each extract at the IC50 of biosynthetic violacein, 0.50 µM.…”

“…Previous work has shown that violacein is capable of killing lab-derived chloroquine-resistant P. falciparum parasites 14 . Here, we showed that patient-sourced clinical isolates, sensitive or resistant to artemisinin, could equally be killed by both commercial and biosynthetic violacein with similar IC50 values.…”

“…Compound mixtures used in the growth inhibition assay consist of mixtures of violacein derivatives (Fig. S4) as described previously 14 .…”

“…Alternative strategies of violacein synthesis are being explored, in particular the use of synthetic biology to engineer industrial bacterial species that can express non-native violacein. Several groups including ours 11 have been successful in implementing a five-gene violacein biosynthetic pathway (vioABCDE) into Escherichia coli or other heterologous hosts 12–14 , providing a route for robust, in-house and inexpensive compound production.…”