Elucidation and derivatisation of bisindole biosynthesis pathways

Abstract

Bisindole natural products encompass the well-studied indolocarbazoles, such as rebeccamycin and staurosporine, among other minor classes including indolotryptolines and bisindolylpyrroles. Bisindole biosynthesis starts from L-tryptophan and forms highly aromatic bisindole scaffolds in a series of biosynthetic steps, which are catalysed by various core scaffold formation and tailoring enzymes that work in tandem to shuttle highly labile bisindole intermediates to the cognate biosynthetic routes. Due to the demonstrated potent biological activities of bisindoles against disease-relevant clinical targets, new bisindoles and their synthetic analogues could yield promising drug candidates with improved activities. This thesis sets out to implement synthetic biology approaches to discover and diversify bisindoles via a two-pronged strategy – (1) by combinatorial assembly of bisindole biosynthesis pathway variants, or in combination with synthetic chemistry to further diversify the chemical scaffolds, and (2) by genome mining of new bisindole biosynthetic gene clusters (BGCs) using existing bioinformatic tools and experimental validation of the BGCs via heterologous expression. The first aim was achieved by derivatising the antimicrobial bisindole violacein scaffold by precursor-directed biosynthesis, in vivo halogenation and semi-synthesis of cross-coupling violacein derivatives based on a brominated scaffold. The violacein biosynthesis pathway was also refactored and heterologously expressed in Escherichia coli as a proof-of-concept demonstration of rapid pathway assembly using the EcoFlex Golden Gate toolkit with user-customisable genetic parts. The second aim was achieved by genome mining for the uniquely conserved chromopyrrolic acid synthase (CPAS) enzyme homologues, which led to the identification of two new bisindole BGCs (acf and nos). Heterologous expression of these BGCs in Streptomyces coelicolor resulted in the discovery of new bisindoles including arcyriaflavin F. Further investigations into new bisindole BGCs with unusual enzymes, in combination with semi-synthetic and synthetic biology approaches, will likely lead to the discovery of novel bisindoles with unprecedented scaffolds and biological activities.