Background: Cell surface display system allows for endowing functional proteins expressed on bacterial surface by fusing different anchor proteins. Among PgsA, Blc, and Omp anchor, the antigen 43 (Ag43)-mediated surface display is a novel system in Escherichia coli. Here, we have demonstrated the red fluorescent protein (RFP) and cellulase (EC 3.2.1.4) on the cell surfaces at two different fusion sites in Ag43. Results: We introduced two fusion sites which are unstructured domain (52-138 aa) and autochaperone domain (600-700 aa) at N-terminal for passenger proteins. As a result, the surface-displayed RFP expressed in plasmid pET28a, but the intracellular RFP expressed more than the surface-displayed RFP. Improved display efficiency of Ag43 was present when fusing at the site of the 138th amino acid (aa) compared to fusing at the site of the 700th aa. For endoglucanase, whole-cell surface-displayed Ag43-138-BsCel5 showed the highest specific activity which was 4.65-fold of BsCel5. Cell-displayed cellulase preserved residual activity ranging from 78% to 38% at temperatures from 55 °C to 80 °C, respectively. Conclusions: This study is to demonstrate the novel surface display system of Ag43 in E. coli by targeting two different proteins RFP and BsCel5 that were successfully displayed on the cell surfaces at two different fusion sites. The Ag43 system displays surface heterologous proteins and is a potential whole-cell catalyst in the bioconversion of cellulose.
Background: Shewanella genus is famous for applications like electron transfer in microbe fuel cells and bioremediation of heavy metals through the Mtr pathway. A potential way to enhance the electron genesis ability of Shewanella is to express exogenous mtr genes via recombinant DNA technology. Thus, to design and develop expression vectors capable of replicating in Shewanella and enhance the genetic toolbox of the same is important. Result: In this study, a plasmid construct with a replication origin, repB, and pLacI promoter is reported for the first time to drive the expression of green fluorescent protein in S. oneidensis MR-1. Based on the same vector, the Mtr pathway genes mtrA, mtrC, and mtrCAB were also successfully expressed in MR-1. The recombinant strains had higher ferric reductase activity compared to the wild type. The highest enzymatic activity of 508.33 U/L in genetic Shewanella with mtrC gene is obtained, which is 1.53-fold higher than that of wild strain. The plasmids were stable up to 90 generations. Conclusion: We have demonstrated an expression system based on pLacI promoter and repB ori in Shewanella. Consequently, the combination of repB and pLacI will have great potential in Shewanella to turn on expression of different genes constitutively.
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