Development Of Bacterial Cell Surface Display Systems Using Ice Nucleation Protein Anchor Towards Biotechnological Applications

The ice nucleation protein (INP), a secretory protein from Pseudomonas syringae, Erwinia sp., and several other Gram-negative bacteria, has drawn a wide attention for development of the bacterial cell surface display systems while used as a functional carrier protein in the recent decade, in term of the expoitation of various biological applications. In this Ph. D dissertation, a novel ice nucleationg gene(inaQ) was cloned from a high ice-nucleating active bacterim, and was characterized of its heterologous expression, followed by the multiple analyses of the outermembrane transport activities of the full-length and truncated expression products, and the cell-surface display efficiencies of the recombinant Escherichia coli or Pseudomonas putida cells that expressing GFP, PBP or AiiA fusion protein whicle using the N-terminal domain of InaQ as the anchoring motif. The work can be summarized as the following:1. Cloning and molecular characterization of inaQTotal DNA of P. syringae MB03 was digested with EcoRI/PstI, the 4-5 kb electrophoresed DNA fragments were purified and then ligated into E. coli cloning vector pRSET-b to allow the construction of a partial gene library. Based on the consensus sequenses of several published inp genes, an oligonucleotide probe was synthezied and used to clone the ice-nucleation protein gene from the libraty via the strategy of colony in situ hybridization. Consequently, a novel ice nucleation protein gene with an integrated encoding frame together with its resident promoter sequences, which was designated as inaQ (GenBank:EU360731), was obtained. Sequence alignment analysis showed that inaQ exhibited a sequence similarity of 89.2%,93.9%and 93.4%, with three previously reported ice nucleation gene inaK, inaV and inaZ genes, respectively, that were cloned from P. syringae. The inaQ gene encoded a protein (InaQ) with 1200 amino acid residues, which can be structurally distinguished as an N-terminal domain (InaQ-N,175 aa), a C-terminal domain (InaQ-C,49 aa) and a central domain with 976 aa. In addition, InaQ-N harboured two tranmembrane segments, whereas InaQ-C harboured more hydrophilic residues.-residue periodicity central repeating domain.2. Expression profile and transmembrane activity of InaQThe acquired ice nucleation activities of E. coli and P. putida recipients by expressing the introduced full-length inaQ were verified. For determination of transmembrane and cell-surface display efficiencies of InaQ, the green fluorescent gene gfp was used as a reporter gene to construct the inaQ/gfp fusion gene, then ligated into NcoI/EcoRI sites of E. coli expression vector pTrcHis-C and expressed upon IPTG induction. The pronase accessibility, SDS and EDTA sensitivity assays were subsequently performed, confirmed that the cell-surface display efficiency of InaQ/GFP fusion protein in E. coli can be over 60%.Although no apparent signal sequences can be predicted, it revealed that a weak transmembrane signal at the position of aa 18 in InaQ by signal peptide prediction analysis. To verify if the first 18 aa segment associates the transmembrane activity of InaQ, a recombinant inaQ-N'/gfp was constructed, at which the first 54 bp sequences was deleted, to allow expressing a truncated InaQ-N'/GFP that lacked the first 18 aa in E. coli construct. The immunofluorescence microscopic observation and FACS analysis indicated that the InaQ-N'/GFP fusion proteins, were expressed in the cytosol but not on the cell surface. Meanwhile, the C-terminal of InaQ was fused to the GFP and expressed in the E. coli constructs. By detecting GFP and Cy5 fluorescence intensity, the fusion proteins failed to immobilize on the cell surface. All these results suggested that the transmembrane activity of InaQ was attributed to its N-terminal domain moiety.3. Construction and analysis of cell surface display system in E. coli and P. putida constructsThe inaQ-N/gfp fusion gene was inserted into the NcoI/EcoRI sites of E. coli JM109 expression vector pTrcHis-C and the shuttle vector pYMBP, respectively. Then the recombinant plasmids were transformed into the E. coli JM109 and P. putida AB92019 and constructed the recombinant strains, respectively. According to the results of immunofluorescence microscopic observation, cell fractionation, Western Blot, pronase accessibility, SDS and EDTA sensitivity assays and FACS analysis, the efficiency of cell surface display on both E. coli and P. putida constructs were over 40%.Then, the 2 and 3 tandemly repeats of inaQ-Ns were further fused to the gfp gene and expressed in E. coli JM109 and P. putida constructs. The cell surface systems were consequently analyzed by cell fractionation, Western Blot, FACS and immunofluorescence microscopic observation. The results indicated that as the number of InaQ-N domain increased, the efficiency of surface display was raising to be more than 60%. Therefore, the synergistic effects of InaQ-N domains had not only improved the expressing efficacy of surface display but also enhanced the ability of outer membrane localization of both recombinant strains.4. Expression of phosphate binding protein on both E. coli and P. putida and the efficiency of phosphate binding ability The phosphate-binding protein (PBP) encoding gene phoS without the signal peptide encoding sequence was cloned from E. coli K-12 by PCR. The inaQ-N/phoS fusion gene was inserted to the vector pTrcHis-C and pYMBP, transformed into host cells, respectively, and expressed. Then, Western Blot, FACS and immunofluorescence microscopic observation analysis were carried out. For the display of InaQ-N/PBP fusions on the surface of both E. coli and P. putida, the capable of absolute biosorption of total phosphates were extremly 80 mg/L and 60 mg/L, respectively, over 5 hours, which were approximately 100-fold and 3-fold compared with control cells. The surface immobilized PBP fusion that had three dandemly repeated InaQ-Ns exhibited the maximum increment in phosphate biosorption (110 mg/L), at sixfold compared with control strain. Even the 42℃heat-killed recombinant cells of either E. coli or P. putida retained substantial biosorptive acitivities.5. Expression of AiiA on the cell surface of P. putida and the efficiency of preventing soft rot diseaseThe AHL-lactonase (AiiA) encoding gene aiiA was cloned from Bacillus thuringiensis BMB171 by PCR. The aiiA gene was digested by BglⅡ/EcoRI and ligated to the same sites of constructed plasmids instead of gfp gene. Then, the recombinant P. putida for expressing inaQ-N/aiiA fusion gene was constructed. The P. putida construct had the function of inhibiting soft rot disease caused by Erwinia carotovora on potato chips. After cell fractionation of P. putida constructs, the cytosol and outer membrane fractions also had ability of inhibiting soft rot disease.Although ice nucleation protein as carrier protein was applied in several areas, the transmembrane domain and secretory mechanisms were still unknown. In this work, the analysis of a new cloned inaQ gene and the expressed InaQ protein indicated that N-teminal domain of InaQ was related to the transmembrane and anchoring function. The result provided evidence for the further researches on secretory mechanisms of ice nucleation protein. Because only the N-terminal of InaQ had the ability of transmembrane and surface localization, the display of heterologous protein on the cell surface of E. coli and P. putida using InaQ-N domain can minimize the molecular weight of anchoring motif and displaying larger passenger proteins. Furthermore, the successful display of phosphate-binding protein resolved the problems for deteriorates of enhanced biological phosphorus removal system under aeration, high carbon and low COD conditions and provided a new and effective method for biosorption of phosphate. The successful display of AHL-lactonase also demonstrated that the constructed cell surface display system gained significant applications in preventing soft rot disease.