The Application Of A Heterologous Cellulase In Recombinant Proteins Production And The Exploration Of The N-terminal Role In Secreting Out Of Escherichia Coli

The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of enzymes and therapeutic proteins, because it is the best characterized host with many available expression and regulation tools. However, the common laboratory strains of E. coli are poor secretors of proteins under normal culture conditions, because this bacterium has a complex cell envelope with two layers. Therefore, heterologous proteins produced in recombinant E. coli are generally intracellular and often in the form of inclusion bodies, from which the biologically active proteins can only be recovered by complicated and costly processes. Extracellular production of heterologous proteins in E. coli will not only provide a simple and convenient production and purification process, but also provide fast and direct screening capabilities for target therapeutic proteins or enzymes that are heterologously expressed in recombinant E. coli.Significant effort to produce target proteins extracellularly in E. coli has been made, with the research efforts split into two categories. (1) Targeted accumulation of the heterologous protein in the periplasmic space through the inner membrane (IM) using a leader peptide, such as PelB, then the heterologous protein is released to the medium through the outer membrane (OM) using cell envelope mutants or lysis proteins; (2) Fusion of the heterologous proteins to fusion partners that can be secreted from the cytosol out of the cells via known or unknown systems.In this study, we report that the catalytic domain of a cellulase (Cel-CD) from Bacillus sp.Z-16 can be efficiently secreted from E. coli when it was heterologously overexpressed. The accumulation of Cel-CD in the culture medium reached 514 mg/L. As the cellulase plays an important role in cellulosic biomass transformation, the extracellular expression of CelCD in E. coli provides a platform for cellulose production. Both the Cel-CD and its N-terminal sequence have potential as fusion partners in the production of various recombinant proteins. We fused a β-glucosidase from Thermobifida fusca YX to Cel-CD. The fusion proteins digested the cellulose outside the host and the resulting glucose could be imported into the cells. Based on this, we set up a PHB production bioprocessing in E. coli strain using cellulose as the sole carbon source.一、The N-terminal role of Cel-CD in secreting out of Escherichia coliIn this part, 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 membrane. This is the first report that a "non-classical signal peptide" can guide recombinant proteins out of the cells from cytoplasm.二、Using Cel-CD as the carrier protein in recombinant protein secretionIn previous study, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. In this part, both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.三、Cel-CD function as a bifunctional proteinIn this part, a β-glucosidase (Tfu0937) was fused to Cel-CD and its N-terminal sequence respectively to obtain E. coli strains that were able to hydrolyze the cellulose. 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 yield of PHB reached 2.57-8.23 wt% content of cell dry weight directly from amorphous cellulose/cellobiose. Moreover, we found the Cel-CD and N20 secretion system can also be used for the extracellular production of other hydrolytic enzymes. This study suggested that a cellulose-utilizing E. coli was created based on a heterologous cellulase secretion system and can be used to produce biofuels and biochemicals directly from cellulose. This system also offers a platform for conversion of other abundant renewable biomass to biofuels and biorefinery products.