From plastics to pathogens: Developing Adhirons as PET-binding modules and inhibitors of bacterial nonulosonic acid biosynthesis

Adhirons are small, non-antibody binding reagents that provide powerful tools for modulating enzyme activity. This thesis explores these binders in two settings. First, their use as novel plastic-binding proteins to augment enzymes that degrade the common polyester, polyethylene terephthalate (PET) - an approach inspired by the auxiliary binding domains that aid in the degradation of natural polymers. Enzymatic depolymerisation offers a sustainable solution to the plastic-waste predicament facing society, however, enzyme-substrate engagement remains challenging. Several PET-binding Adhirons were characterised across powder and film substrates using a mixture of biochemical approaches, with affinities explored via binding isotherms. Adhiron contributions to PET hydrolysis were interrogated via the generation of an IsPETase–Adhiron fusion library and a high-throughput pipeline that allowed quantification of PET depolymerisation via RP-HPLC directly from cell lysates. The identification and characterisation of multiple fusion candidates revealed insights into plastic–protein interactions and the overall limitations of this approach for improving catalysis. Secondly, Adhirons were pursued as inhibitors of bacterial sugar production - namely, pseudaminic acid (Pse) and legionaminic acid (Leg) - two nonulosonic acids associated with virulence in a number of pathogenic species. A high-resolution structure of CMP-pseudaminic acid synthetase (PseF) in complex with its product, CMP-Pse, provided structural background for understanding the potential inhibition of this enzyme and its homologue LegF. Phage display was used to screen Adhirons against both enzymes, with candidates selected through a combination of sequence clustering and complex modelling, and binding subsequently characterised via a thermal shift assay. Several PseF binders exhibited an activating effect and these were further explored through co-purification and native mass spectrometry. Though contrary to the original aim, activators may prove useful in other biotechnological contexts and provide a route to better understand the conformational changes associated with these enzymes.