Establishing structure-led design rules for spore surface-display of enzymes of commercial interest

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Bacterial (endo)spores, formed by Bacillales and Clostridiales members when nutrient starved, are the most durable cells found in nature. The composition and assembly of spores is well understood at genetic and biochemical levels, but less so at the molecular structural level. This knowledge gap is apparent in current approaches to spore surface display - molecular genetic manipulation of typically Bacillus subtilis to express and deposit heterologous proteins on the outermost spore coat. So far, only a handful of coat proteins have been successful fusion partners for heterologous proteins of interest, despite the coat containing over 70 distinct proteins. Current surface display methodology relies on trial and error, which is time consuming and often ends in failure. As such, it has failed to translate to the commercial sphere that its potential benefits merit. Recently, the structural landscape has changed through advances in successful crystallisation of spore proteins and by the generation of seemingly accurate structural models by AlphaFold. This projectaims to use this information to establish structure-led design principles that more consistently result in successful display of functional proteins on or within the spore coat. The project will be conducted with the support of Johnson Matthey and will focus on the display of enzymes of commercial interest and examining the behaviour of cells intended for spore surface display as they transition from lab to pilot scale.