Fig. 2

Applications of B. subtilis surface display technology in microbial cell bioprocessing: A B. subtilis surface display technology for biocatalyst development. A specialized E. coli–B. subtilis shuttle expression vector, pHS-cotG-lip, was engineered for LiP display on the B. subtilis spore surface. The lip gene was fused to the CotG gene, an outer spore coat protein, facilitating stable anchoring of the LiP protein. The expression of the fusion construct was driven by a strong sporulation-specific promoter, ensuring optimal production during the sporulation phase. Following sporulation, the LiP protein was successfully displayed on the spore surface. B B. subtilis surface display technology for bioremediation. The first step is to select a metal-binding protein that can specifically interact with and bind to toxic metals, such as cadmium or mercury. These proteins typically have a high affinity for specific metal ions, making them ideal for bioremediation applications. The plasmid is engineered to include the cell wall anchoring domain from a putative sortase substrate, YhcR at the C-terminal of the metal-binding protein. Upon successful transformation, Sortase A recognizes and cleaves the sorting signal and catalyzes the attachment of the metal-binding protein to the bacterial cell wall. Once anchored to the surface of B. subtilis, the metal-binding protein binds to toxic metals, such as cadmium, from contaminated environments. This enables the bacteria to efficiently adsorb and sequester the harmful metals. C B. subtilis surface display for biosensor development. The plasmid is designed to express both the biosensor protein (such as arsenic-binding protein) and a reporter protein, GFP (green fluorescent protein). The GFP gene is included in the plasmid as a visual marker for detecting biosensor activity. The plasmid also contains a sequence for an anchoring protein, such as CotB, to ensure the surface display of the biosensor protein on B. subtilis spores. After the plasmid is introduced into B. subtilis through transformation, the cells are induced to undergo sporulation. During this process, the biosensor (arsenic-binding protein) is displayed on the surface of the spores, while GFP is expressed inside the cells. Upon exposure to arsenic(III) or another target contaminant, the biosensor protein binds to the target molecule. This binding can trigger a conformational change or activate GFP expression, making the biosensor detectable. The activation of GFP, in response to the binding of arsenic(III) or the target molecule, can be monitored using fluorescence. The intensity of the green fluorescence correlates with the amount of arsenic(III) in the sample, enabling real-time detection