Advancing protein display on bacterial spores through an extensive survey of coat components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SP investigated, 26 formed functional fusions with β-glucuronidase (GUS) - a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed the 1) SP promoter activation dynamics during growth, 2) supported GUS activity on spores, 3) surface availability, 4) protection from thermal and proteolytic degradation, and 5) compatibility of co-expression of many of these fusions. Multi-copy expression and pairwise co-expression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and difficulty in predicting compatibility between different SPs fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA ( s mall s pore c oat assembly protein A ) as an effective spore display carrier. SscA supported enzyme activity at least fourfold higher than any other SP, including the well-established and popular anchor, CotY. We attribute this to the biochemical and genetic features of SscA; its promoter demonstrated early and sustained activation relative to other SPs and its small size (∼3 KDa) likely minimally interferes with enzyme folding, oligomerization, and activity. Although the specific localization of SscA within the spore coat has not been established, its hydrophobic nature and low surface availability suggests that it assembles internally within the spore coat, which makes it highly stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledgebase to advance the biotechnological utility of B. subtilis spores.