Orchestrating an Enzyme Ensemble Towards Novel Antibiotics

Violacein is a violet pigment first isolated from Chromobacterium violaceum and is part of the indolocarbazole biosynthetic family that utilises L-tryptophan as the starting substrate. The violacein pathway is encoded within a conserved operon of 5 genes (vioABCDE), whose gene-products catalyse a 14-electron oxidative biosynthesis pathway[1]. The indolocarbazole biosynthetic pathways have attracted immense interests because of their therapeutic potential for medical applications, including antimicrobial, antiviral, trypanocidal and anti-tumorigenic properties[2].

The violacein pathway is a favourable model pathway in synthetic biology for combinatorial biosynthesis because of its relative simplicity and production of coloured intermediates, which allows high-throughput screening. Recent study into the effect of promoter strength[3] on violacein yield have indicated that heterologous production of violacein has the potential to surpass natural supply. In my project, I have implemented a combinatorial library screening method for violacein production using EcoFlex, a Golden Gate DNA assembly kit developed by our lab (in review). The library variants, which differ by their promoter, RBS and terminator sequences at each of the three selected violacein pathway genes, exhibited a wide range of violacein yields with the highest producing strain at 66 mg L^-1. It was also found that low-copy plasmid number was better suited for library construction for toxic pathways like violacein, as observed by the decrease in frequency of mutation in the gene regulatory parts. In the presence of strong selective pressure (toxic pathway), the cells will likely to mutate the gene regulatory components to increase survivability.

As a separate story, due to the homology of enzymes involved in the indolocarbazole biosynthetic pathways, some researchers have attempted to create novel metabolites using a combination of enzymes involved in different pathways[4]. It is envisaged that the diversification of metabolites will lead to discovery of novel compounds, which may exhibit therapeutic effect better than violacein upon screening against a wide range of disease biomarkers. Potentially synthetic biology can be used to incorporate site-specific labelling enzymes for halogenation, methylation and hydroxylation to create synthetic analogues of violacein, possibly via homologous enzymes from related indolocarbazole pathways or through rational structure-based engineering. To begin, we have crystallised and solved the structure of a tryptophan-bound VioA, FAD-dependent L-tryptophan oxidase, showing the close proximity of the tryptophan substrate with FAD cofactor in a 2.6 Å crystal structure. Using the structure of VioA, we will attempt to rationally engineer VioA to accept tryptophan derivative as a substrate, thus paving the way towards biosynthesis of violacein analogues.

References
1. Balibar, C. J. & Walsh, C. T. In Vitro Biosynthesis of Violacein from L -Tryptophan by the Enzymes VioA - E from Chromobacterium V iolaceum †. 15444–15457 (2006).
2. Durán, M. et al. Potential applications of violacein: a microbial pigment. Med. Chem. Res. 21, 1524–1532 (2011).
3. Jones, J. A. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci. Rep. 5, 11301 (2015).
4. Du, Y.-L. & Ryan, K. S. Expansion of Bisindole Biosynthetic Pathways by Combinatorial Construction. ACS Synth. Biol. 150102122128000 (2015). doi:10.1021/sb5003218