We conducted microbial screening of the local selenium-metal mining area in Lanzhou and selected several six potential reducing competent strains with high efficiency. We simultaneously tested its reduction ability to prove tetravalent selenium and explored the possible proteins acting in it, setting three parallel actions as controls. We then used bioinformatics to find the functional reductase SerV01, which can efficiently reduce selenite in the most reducing bacteria LZ-01(S. aureus), exploring its homology, enzymatic activity mechanics and ligands. The N-terminal domain sequence of ice cryonuclease, the carrier protein we chose, was fused with the INP-N-SerV01 peptide of the bioase reductant SerV01.
The stability of the passenger protein derived from microorganisms is affected by operating conditions during direct extraction and synthesis, and also causes huge economic costs. Therefore, for the purpose of the experiment, we modified the passenger protein to achieve the overexpression of homologous or heterologous proteins through the transformation of high-copy plasmids. This method not only reduces the cost, but also increases the utilization rate of passenger protein. The cell vector contains multiple cryptic plasmids that may have effects on subsequent live expression of enzymes. We tested that the plasmids pSB1A3 from iGEM data resource could be successfully converted into EcN, and we insert strong promoter and long gene sequence as intergenic region to ensure subsequent high quality overexpression of protein (2018IGEM Distribution Kit, containing the constitutive promoter Plac). In our project, ice riboprotein(INP-N) was specifically used to anchor the target genetoto form the fusion polypeptide INP-SerV01 that can effectively anchor on the cell outer membrane.
Because intestinal microorganisms usually play an important role in the REDOX of substrates and can lead to changes in metal valence states during the adsorption and detoxification of heavy metals, while among them, EcN, a type of non-pathogenic symbiotic gram-negative probiotics, has a good probiotic effect and is used for the treatment of various intestinal diseases because it does not secrete exotoxin. Moreover, it is the host strain with the dominant expression of foreign protein. Analysis of EcN’s genomic structure suggests that it lacks virulence factors such as α-hemolystin, P fiber adhesins and semi-crude lipopolysaccharide phenotypes. Its expression systems containing suitable factors (e. g., microtin, adhesin and iron absorption) contributes to the properties of its probiotical traits.
Thus we chose this strain as our cell vector. We then anchored to the extracellular membrane of probiotics EcN to construct whole cell biocatalyst EcN-IS, which finally reduced selenite on the surface of the cell membrane in the pariplasmic space by applying the Microbial Cell Surface Display System, a system that acts on the surface of the cell membrane or cell wall of the receptor microorganism by combining specific foreign functional proteins with anchorin. Fusion proteins can be directly expressed on the surface of host cells for practical application purposes and are often used to demonstrate the adsorption and removal of excessive and excessive heavy metals by metal binding proteins. Cell-surface display technology can also be used to enhance the stability of catalytic hydrolases to resist temperature changes, and the fusion proteins of surface display as catalysts can be recycled, which is cost-effective compared to the high recovery cost of immobilized enzymes.