Construction and Mechanism Exploration of Highly Efficient System for Bacterial Ghosts Preparation Based on Engineered Phage ID52 Lysis Protein E

Abstract

Bacterial ghosts (BGs) are hollow bacterial cell envelopes with intact cellular structures, presenting as promising candidates for various biotechnological and biomedical applications. However, the yield and productivity of BGs have encountered limitations, hindering their large-scale preparation and multi-faceted applications of BGs. Further optimization of BGs is needed for the commercial application of BG technology. In this study, we screened out the most effective lysis protein ID52-E-W4A among 13 mutants based on phage ID52 lysis protein E and optimized the liquid culture medium for preparing Escherichia coli Nissle 1917 (EcN). The results revealed a significantly higher lysis rate of ID52-E-W4A compared to that of ID52-E in the 2xYT medium. Furthermore, EcN BGs were cultivated in a fermenter, achieving an initial OD600 as high as 6.0 after optimization, indicating enhanced BG production. Moreover, the yield of ID52-E-W4A-induced BGs reached 67.0%, contrasting with only a 3.1% yield from φX174-E-induced BGs. The extended applicability of the lysis protein ID52-E-W4A was demonstrated through the preparation of Salmonella pullorum ghosts and Salmonella choleraesuis ghosts. Knocking out the molecular chaperone gene slyD and dnaJ revealed that ID52-mediated BGs could still undergo lysis. Conversely, overexpression of integral membrane enzyme gene mraY resulted in the loss of lysis activity for ID52-E, suggesting that the lysis protein ID52-E may no longer rely on SlyD or DnaJ to function, with MraY potentially being the target of ID52-E. This study introduces a novel approach utilizing ID52-E-W4A for recombinant expression, accelerating the BG formation and thereby enhancing BG yield and productivity.