Development of a bioreactor system using Bacillus subtilis spores
Daisuke IMAMURA
Hosei University, Faculty of Bioscience and Applied Chemistry
Report:To develop a system that utilizes Bacillus subtilis spores that display useful proteins on
their surfaces as bioreactor particles called sporobeads, I first localized spore coat proteins inthe spore layers and identified proteins located in the outermost layer of the spore coat. Next,
I measured fluorescence intensities of the spores with green fluorescent protein (GFP) fusedwith these spore coat proteins to assess their abundance and stability on the spore surface.
Four proteins were found to be able to display exogenous protein at both 4 ºC and room
temperature (20-25 ºC), under wet and dry conditions for at least 3 weeks. Furthermore,disruption of a gene essential for germination, cwlD, did not affect the stability of proteinsdisplayed on the spore surface. These results indicate that these 4 proteins are promising
candidates for anchor proteins in this sporobead system.
Research aims
Displaying exogenous proteins or peptides on the surface of small particles is useful inmany fields, including medical science, pharmaceutical science, bioremediation, thefermentation industry, and macromolecular material production. Efforts to develop suchdisplay systems on the surface of bacterial, mammalian, and insect cells have been reported.
This research aims to develop a display system using B. subtilis spores. B. subtilis spores
have many advantageous features for their utilization for this purpose. 1) B. subtilis is safefor humans, as B. subtilis subsp. natto is known to be used for natto production. 2) Easy andlow-cost incubation and knowledge of the natto industry can be applied. 3) Since B. subtilis
spores are highly resistant to harsh environments, they can be used under a wide range ofconditions and are amenable to long storage or transportation. In addition, it is known that
fixed enzymes are more stable than those free in solution. 4) To display proteins normally
expressed in a cell on a cell surface, the protein must clear several hurdles including
secretion efficiency, attachment to the membrane, and refolding after secretion. B. subtilis
spores do not face these problems, because spore coat proteins are expressed in the mothercell and assembled on the forespore (Fig. 1). Therefore, this system is useful for a wide
variety of purposes and in many reaction conditions. This research aims to identify the bestanchoring protein to establish a protein displaying system with B. subtilis spores.

Methods
It is necessary to identify proteins
exposed on the spore surface fromamong 70+ spore coat proteins, toestablish a protein display system using
B. subtilis spores (Fig. 1). To date,
localization of each protein within thespore coat layer has only beenaccomplished using immuneelectron
microscopy, and the results continue tobe poorly understood. Therefore, wedeveloped a method to identify theposition of each spore coat protein
within the spore coat layers using
fluorescence microscopy combinedwith image analysis and statisticalprocessing 1). Using this technique, we
discovered the previously unidentified
outermost spore layer 2, 3). We analyzed
the location of several spore coat proteins and chose several proteins found on the spore
surface as candidate anchoring proteins on which to display enzymes. Fluorescenceintensities of spores expressing GFP-surface protein fusions were measured to assess theabundance and stability of displayed proteins. One potential flaw in this system is that if thesubstrate or products of a bioreactor reaction induce spore germination, theenzyme-displaying particles will be lost. To eliminate this possibility, we disrupted a geneessential for spore germination, cwlD, and assessed the abundance and stability of displayedprotein on these mutant spores to identify the best anchor protein.
Results
Localization analysis of spore coat proteins in the spore layers identified 6 proteins at theoutermost layer of the spore. We then assessed the abundance and stability of GFP anchoredto these proteins on spores. Four proteins, CgeA, CotW, CotX and CotZ, were found todisplay GFP stably and abundantly at 4 ºC as well as room temperature, in wet and dryconditions. Fig. 2 shows the data for dry conditions. Although each anchor protein exhibiteda different initial fluorescence intensity, all proteins retained at least half of their initial
fluorescence after 23 days of storage at 4 ºC or room temperature (Fig. 2). These results
indicate that CgeA, CotW, CotX, and CotZ can display exogenous protein on the surface ofspores for at least a few weeks. In addition, since there was no significant difference ineffects of the temperature (i.e., 4 ºC and room temperature) on the ability to display proteins,
this system can retain enzymes stably on spores, without low temperature storage.

This spore display system can be used in bioreactors by fusing enzymes required for areaction to these anchoring proteins. However, if the substrate or reaction product induces
spore germination, the enzyme particles would be lost. Thus, we tested whether GFP isstably displayed on spores lacking an essential gene for spore germination. GFP fused to fourproteins was retained at equivalent levels to that on wild type spores and on spores lacking
an essential germination gene.
Taken together, the above results indicate that four proteins identified in this study, CgeA,CotW, CotX, and CotZ, can display exogenous proteins for several weeks at room
temperature, and spores lacking a germination gene were also stable.
Conclusion
This research identified four spore coat proteins that are suitable for long term display ofexogenous proteins on the surface of spores. Thus, these proteins are promising candidate
anchoring proteins for use of spores in bioreactors.
References
1) Imamura, D., Kuwana, R., Takamatsu, H. and Watabe, K. (2010) Localization of proteinsto different layers and regions of Bacillus subtilis spore coats. J. Bacteriol. 192: 518-524.
2) Imamura, D., Kuwana, R., Takamatsu, H., and Watabe, K. (2011) Proteins involved information of the outermost layer of Bacillus subtilis spores. J. Bacteriol. 193: 4075-4080.
3) Shuster, B. et al. (2019) Expansion of the Spore Surface Polysaccharide Layer in Bacillus
subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification
Enzymes. J. Bacteriol. 201: e00321-19.