Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin−Dockerin Interactions

Abstract

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme β-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh–Doc based spore display can hydrolyze o-nitrophenyl-β-D-galactopyranoside. Further, the optimized Coh–Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh–Doc interaction, which will find application in improving the efficiency of B. subtilis spore display.

Mechanisms and Applications of Bacterial Sporulation and Germination in the Intestine

“…This binding is already limited by low binding properties and low protein expression levels in the spore. However, workarounds such as systems utilizing cellulosome-derived cohesin–dockerin interactions to increase the binding affinity of the target proteins to spore surface anchors, have improved efficiency [ 225 ]. Recent developments in spore engineering have suggested genetic alteration of the spore coat proteins to induce additional bacterial adhesion molecules, encouraging stronger epithelial binding [ 226 ].…”

Emerging enzyme surface display systems for waste resource recovery

“…Under adverse environmental conditions, some bacterial species undergo sporulation to produce highly resistant dormant spores through asymmetric cell division that are then released from the mother cell on lysis (Hwang et al, 2011 ; Lin et al, 2020 ; Wang, Jiang, et al, 2021 ). Bacillus subtilis is a well‐characterized model organism, and its spores contain an array of coat proteins that can tolerate recombinant insertions (Lin et al, 2020 ; Wang, Jiang, et al, 2021 ).…”

“…Under adverse environmental conditions, some bacterial species undergo sporulation to produce highly resistant dormant spores through asymmetric cell division that are then released from the mother cell on lysis (Hwang et al, 2011 ; Lin et al, 2020 ; Wang, Jiang, et al, 2021 ). Bacillus subtilis is a well‐characterized model organism, and its spores contain an array of coat proteins that can tolerate recombinant insertions (Lin et al, 2020 ; Wang, Jiang, et al, 2021 ). Bacillus subtilis spore surface display (BSSD) is a recently developed CSD alternative in which mother cells are engineered to produce spore coat‐fusion proteins to display heterologous domains on spore surfaces during sporulation (Hwang et al, 2011 ; Isticato et al, 2001 ; Lin et al, 2020 ; Wang, Jiang, et al, 2021 ) (Figure 2 ).…”

“…Bacillus subtilis is a well‐characterized model organism, and its spores contain an array of coat proteins that can tolerate recombinant insertions (Lin et al, 2020 ; Wang, Jiang, et al, 2021 ). Bacillus subtilis spore surface display (BSSD) is a recently developed CSD alternative in which mother cells are engineered to produce spore coat‐fusion proteins to display heterologous domains on spore surfaces during sporulation (Hwang et al, 2011 ; Isticato et al, 2001 ; Lin et al, 2020 ; Wang, Jiang, et al, 2021 ) (Figure 2 ). Spore ability to survive harsh conditions (due to their multiple, thick coat layers), easy purification, and generally‐recognized‐as‐safe (GRAS) designation, makes BSSD particularly useful for industrial applications (Chen, Ullah, & Jia, 2017 ; Cho et al, 2011 ).…”

Enzyme Immobilization

“…In the first example, the enzyme is immobilized and stabilized by immobilization on gold nanoparticles modified with polyvinyl alcohol [ 20 ]. Another paper shows the β-galactosidase immobilization on a Bacillus subtilis spore [ 21 ]. The authors introduce the spore divergent cohesin modules that can specifically bind to the target enzyme bearing the matching dockerins.…”