Directed evolution of bioactive compounds: Azol(in)e-containing post-translationally modified peptides

Thiazole/oxazole-modified microcins (TOMMs) are a class of post-translationally modified peptides, found in Bacteria and Archaea, and shown to have a wide range of biological activities, including antimicrobial, anti-cancer and anti-malarial compounds. While many TOMMs have been isolated from natural sources and some chemically synthesised, this class of peptide compound remains an underexplored potential source for novel bioactive molecules: chemical synthesis of polyazol(in)es is challenging, while in vivo synthesis relies on heteromeric protein complexes that have not been extensively characterised. The goal of the research project is to establish the E. coli microcin B17 synthase as a platform of discovery and production of novel microcins with diverse function. To achieve that goal, the PhD project will focus on two areas: characterisation of the E. coli microcin B17 complex and the directed evolution of novel microcins. The Pinheiro group has recently established an in vivo platform for the heterologous expression of E. coli microcin B17 precursor (McbA) and synthase, as well as a functional screen based on the co-culture of microcin producer and target strains. The platform has a screening capacity between 103~105 variants per assay. Initially, the project will explore the functional space around the B17 synthase subunits, targeting mutations to predicted functional residues. The residues have already been identified by the Pinheiro group and mutations will be introduced by Darwin Assembly. Synthetase variants will be screened for function and purified synthetase components analysed for stability and structure. The second part of the project will focus on the isolation of novel microcins from a library sampling the sequence neighbourhood (both composition and length) of the wild-type B17. To efficiently explore the sequence space, libraries will be assembled using InDel assembly (a recently developed strategy by the Pinheiro group for the efficient exploration of the sequence space across different lengths. The library will be screened against a range of potential target organisms (e.g. B. subtilis, M. luteus, P. putida, E. coli) and active variants characterised for host range and potency. Another component of this part of the project will be the further optimisation of the InDel assembly platform, integrating it to automated liquid handling platforms and exploring ways to increase the efficiency of the assembly cycle.