Abstract
Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep- etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. QFDE-EM analysis also revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan pores in wild-type E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as outer-inner membrane vesicles (OIMVs). Meanwhile, the secretion of recombinant GFP expressed in the cytosol of the ΔmlaEΔnlpI cells was about 2mg/L which was more than 100 times higher than that of WT, suggesting that OMV phenomenon is able to be applied for the secretory protein expression system of E. coli. Next, secretory production of fatty acid via OMVs was also examined using the multiple gene knockout mutant strain. However, it was failed due to the sever suppression of cell growth and fatty acid production. Expression suppression of mlaE and nlpI using Clustered Regularly Interspaced Palindromic Repeats Interference (CRISPRi) achieved the increase of OMV production while maintaining cell activity. In the future, we will optimize the expression levels of each gene for secrete production of useful compounds via OMVs.
