Directed evolution of bioactive compounds: oxa(thia)zole-containing post-translationally modified peptides

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

Selection platforms for directed evolution in synthetic biology

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules – gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function – be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code