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. In previous report, we identified that the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis can be secreted into the medium from recombinant E. coli in large quantities without its native signal peptide. In this study, we proved the N-terminal sequence of the full length Cel-CD played a crucial role in transportation through both inner and outer membranes. 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. Biotechnol. Bioeng. 2016;113: 2561-2567. © 2016 Wiley Periodicals, Inc.
Keywords: Escherichia coli; heterologous signal sequence; random mutagenesis; secretion system.
© 2016 Wiley Periodicals, Inc.