An extracellular protein expression system in Escherichia coli implies potential application
September 24-28, 2017
Escherichia coli is commonly used as a host for the extracellular production of proteins. However, its secretion capacity is often limited to a frustratingly low level compared with other expression hosts, because E. coli has a complex cell envelope with two layers. We recently identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli in large quantities without its native signal peptide. By subcellular location analysis, we verified that the secretion was a two-step process via the SecB-dependent pathway through the inner membrane and an unknown pathway through the outer membrane. However, the N-terminal region of Cel-CD is polar and hydrophilic, which showed no similarities to other typical signal sequences. Random mutagenesis experiment suggested that the N-terminal sequence is a compromising result of transportation through inner and outer membranes. This is the first report that a "non-classical signal peptide" can guide recombinant proteins out of the cells from cytoplasm. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins with a concentration from 101 to 691 mg/L in flask cultivation.
This protein can degrading cellulose efficiently in the culture medium indicating a great potential. Therefore, a recombinant E.coli that can directly utilize cellulose as sole carbon source by fusion Cel-CD with a b-glucosidase was constructed. Recombinant strains were confirmed to use the amorphous cellulose as well as cellobiose as the sole carbon source for growth. Furthermore, both strains were engineered with poly (3-hydroxybutyrate) (PHB) synthesis pathway to demonstrate the production of biodegradable polyesters directly from cellulose materials without exogenously added cellulases. The results suggested that this system has a potential application in lignocellulosic biomass degradation and biochemical biofuel production.
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Qingsheng Qi, Dongfang Gao, Quanfeng Liang, and Junshu Wang, "An extracellular protein expression system in Escherichia coli implies potential application" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/enzyme_xxiv/51