Construction Of Cell Surface Display System In Bacillus Sp.and Application In Bioremediation Of Environmental Pollution

The microbial surface display technology is a powerful method to display the protein on the cell surface and has many advantages in the fields of agriculture,industry and bioremediation.Bacillus subtilis isan environmentally friendly and safe modal Gram-positive bacterium,many anchoring proteins have been identified and used to display the target proteins on their spores.But few anchoring proteins on the bacterial cell surface were discovered as the cell display systems.Therefore,developing an optimized and efficient microbial cell surface display system is of great significance for practical applications.In this study,we constructed a systematical bioinformatics protocol to screen for the potential anchoring proteins from Bacillus subtilis.As a result,47 candidate anchored proteins and its origin promoters were screened by the method.A EGFP fluorescence reporter system was constructed to assess the cell display efficiency of selected membrane proteins and their promoters.And the anchoring protein SpoIIIJ demonstrated the strongest anchoring activity of all the selected anchoring proteins from Bacillus subtilis.In order to determine the location of the expressed fusion protein,the expression of the fusion protein in the microbial cells was firstly determined using the spore culture medium and the nutrient rich medium,and then the location of the fusion protein which on the cell surface was confirmed by super-resolution confocal microscopy A1/SIM/STORM.A linker,6 prolines,was designed to link the anchoring protein SpoIIIJ and EGFP,which could increase the expression of the fusion protein by 58.32%.Then,a more efficient surface display system was developed in Bacillus licheniformis using the selected anchoring protein SpoIIIJ.Organophosphorus hydrolase OphC2 was then displayed on the surface of the B.subtilis and B.licheniformis systems,respectively.The result showed that the fusion protein was normally expressed on the cell surface.And its activity was 2.1 times more in B.licheniformis than that in B.subtilis.When the metal-binding protein CadR was displayed on the cell surface of Bacillus subtilis,the amount of adsorbing cadmium ions was nearly 7 times higher than that of the original strain.When catalase Cat9 was expressed in Bacillus subtilis,the recombinant cells had enzyme activity,and the SDS-PAGE results further showed that the target band of the fusion protein was more obvious in the cells than in supernatant,which was consistent with the results of fluorescence localization experiments.Therefore,our results indicated that the cell surface expression system can be used to display target proteins on cells and provided great possibilities for bioremediation of environmental pollution.This surface display system will be practically applied to pesticide degradation,bio-catalysis,bioremediation and biosensing.