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

细菌(Endo)孢子是自然界中发现的最持久的细胞，由芽孢杆菌和梭状芽孢杆菌成员在营养匮乏时形成。孢子的组成和组装在遗传和生化水平上是很清楚的，但在分子结构水平上就不那么清楚了。这一知识差距在目前的孢子表面展示方法中是显而易见的--对典型的枯草芽孢杆菌进行分子遗传操作，在最外层的孢子皮上表达和沉积异源蛋白。到目前为止，尽管外壳含有70多种不同的蛋白质，但只有少数外壳蛋白成功地与目标异源蛋白质融合。目前的表面显示方法依赖于反复试验，这很耗时，而且往往以失败告终。因此，它未能向商业领域转化为其潜在好处的价值。最近，由于孢子蛋白质的成功结晶和AlphaFold生成看似准确的结构模型的进展，结构格局发生了变化。本项目旨在利用这些信息来建立结构导向的设计原则，从而更一致地在孢子被上或内成功展示功能蛋白质。该项目将在Johnson Matthee的支持下进行，重点是展示具有商业价值的酶，并检查拟用于孢子表面展示的细胞从实验室过渡到中试规模时的行为。

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.