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Construction of hypervesiculation Escherichia coli strains and application for secretory protein production
Author(s) -
Ojima Yoshihiro,
Sawabe Tomomi,
Konami Katsuya,
Azuma Masayuki
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27239
Subject(s) - bacterial outer membrane , secretion , escherichia coli , green fluorescent protein , western blot , secretory protein , biology , mutant , gel electrophoresis , bacteria , microbiology and biotechnology , vesicle , extracellular , enterobacteriaceae , biochemistry , gene , membrane , genetics
Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram‐negative bacteria, including Escherichia coli . Several gene‐deficient mutants relating to envelope stress ( nlpI and degP ) and phospholipid accumulation in the outer leaflet of the outer membrane ( mlaA and mlaE ) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double‐gene‐knockout mutant (Δ mlaE Δ nlpI ) showed the highest OMV production. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis‐based quantitative analysis showed that OMV production by strain Δ mlaE Δ nlpI was ~30 times that by the wild‐type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain Δ mlaE Δ nlpI/gfp , 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.