Construction And Properties Of Specific Lead-binding Protein Surface Displaying Bacteria

Recently,the application of cell surface display has become the research focus in the field of heavy metal repair.In this study,the N-terminal sequence of ice-nucleation protein(INPN)was selected as the anchor protein of the outer membrane of the cell.First,a series of enhanced green fluorescent protein surface display expression system was constructed in E.coli to investigate the transport and anchoring function of INPN.And the effect of fusion pattern between the anchor protein and the target protein and the culture temperature on the fluorescence expression of engineered bacteria in this system was studied.The results show that INPN can be used as a carrier protein in E.coli cells,and the fluorescence expression of engineered bacteria is greatly affected by the fusion pattern.Inappropriate linker peptide may cause the target protein to misfold.Studies on the induction temperature found that 22?is the most favorable temperature for protein expression.Based on the above work,through the same fusion pattern,the two transcription regulators PbrR and PbrR691 and the lead storage protein PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli cells,with only the N-domain of ice-nucleation protein as an anchor protein to achieve specific adsorption of lead ions(Pb²⁺)and bioremediation of lead pollution;the specific localization of fusion proteins on the outer membrane was confirmed by cell fractionation,western bloting analysis,and whole-cell immunofluorescence.We also investigated the effects of fusion pattern,the expression level of fusion protein controlled by the concentration of inducer directly,heavy metal concentration,and heavy metal type on the adsorption of Pb²⁺by these engineered bacteria,and the optimal linker peptide(flexible linker)and inducer concentration(0.5 m M)were obtained.The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb²⁺.Under the interference of other heavy metal ions,the adsorption capacity of engineered bacteria for lead has reached 5 times higher than cadmium and12 times higher than mercury;and the maximum Pb²⁺adsorption amounts of the three proteins(PbrR,PbrR691,and PbrD)were 942.1,754.3,and 864.9?mol/g cell dry weight,respectively,which are the highest lead adsorption amounts of these three proteins reported to date.To confirm the actual detoxification effect of the engineered bacteria constructed in this study,a bioassay method based on the Nicotiana benthamiana plant was established.The engineered E.coli bacteria were applied to seed germination and growth of Nicotiana benthamiana in a lead-contaminated environment,and clear detoxification effects were observed.The seed germination rates treated with PbrR,PbrR691 and PbrD surface displaying strains were 2.5,2.7,and 2.4 times higher than that of the control group,and the average bud length reached 8.5,7.9,and 9 times than that of the control group respectively;in the soil detoxification test,the total plant biomass under the treatment of the three engineered bacteria reached 7.45,13.35,and 7.3 times of the wild type strain BL21,indicating that the detoxification effects of the engineered bacteria are obvious and can be used as cell adsorbents that specifically binds lead ions.In summary,the PbrR,PbrR691,and PbrD surface displaying bacteria designed and constructed in this study have high adsorption capacity,selective specificity,and good application value for Pb²⁺,which may provide a convincing basis for in situ remediation of lead-contaminated water or soil.