Tizei, Pedro A.G.; (2018) Directed evolution of bioactive compounds: oxa(thia)zole-containing post-translationally modified peptides. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Thiazole/Oxazole Modified Microcins (TOMMs) are a diverse class of post-translationally modified peptides including many bioactive compounds; a potential new source for drug discovery. Despite a limited understanding of the TOMM synthase heterotrimeric complex biosynthetic mechanism, a variable degree of substrate plasticity is present in the family. This makes them attractive targets for developing novel oxazole- and thiazole-containing compounds from synthetic peptides. Available annotation on complex members suggests the presence of different biochemical activities among homologous proteins, precluding the use of established prediction methods for identification of functional residues. A novel algorithm was developed (Normalised Shannon Entropy, NoSE) for functional prediction from sequence alignments containing mixed functions. NoSE was applied, along with established conservation- and coevolution-based metrics, to detect functional residues in the well-characterised bacterial Solute Binding Protein family, which could be validated against the extensively reported characterisation. The strategy was applied for functional residue prediction in the TOMM synthase complex and candidate functional residues were mutated in McbC dehydrogenase of Escherichia coli. Mutants were assessed using a bacterial growth inhibition bioassay and six out of sixteen mutations reduced TOMM production, demonstrating the value of employing a prediction strategy to improve characterization of proteins. Attempts at establishing an in vitro assay for TOMM biosynthesis were unsuccessful due to difficulties in protein expression and purification, as well as inconsistent assay results. Finally, a framework for directed evolution of length-variable proteins was developed, with the aim of engineering synthetic TOMM products. A method was developed for assembly of high-quality libraries at a low cost, along with a workflow for enriched motif detection in selection experiments. The approach was validated by isolating seven novel variants of the β-lactamase TEM-1 active on a non-cognate substrate. Together, the developed methods represent a foundation for establishing TOMM biosynthesis as a platform for discovery of novel bioactive compounds.

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