Exploring The Translocation Mechanism Of Endoglucanase BcsZ In Escherichia Coli

Endocellulase plays a prominent role in biochemistry industries such as medicine,food and biofuels.It is well established that they are secreted to the extracellular from many different types of organisms;however,few studies have focused on their secretory mechanisms.Previous studies have revealed that endoglucanase BcsZ can be secreted to the outside of E.coli DH5a cells,but its secretion mechanism remains ignorant.Therefore,we have studying on BcsZ translocation.We demonstrated that extracellular accumulation of BcsZ is a specific secretory activity,which is not caused by E.coli cell lysis.In addition,secretion of BcsZ is a two-step process via the periplasmic space.A typical signal peptide consisting of 21-amino acid residues at the N-terminus of BcsZ is indispensable for transport,but we suggested that the Sec and TAT pathways do not participate in BcsZ export across the inner membrane.It is high likely that there would be some unknown porins that mediate BcsZ transmembrane transport.At the same time,a preliminary exploration of BcsZ translocation crossing E.coli outer membrane was conducted,but without a clear conclusion.To explore the specific mechanism of BcsZ exporting across the inner membrane.we developed a growth-coupled screening strategy to find genes that affect the secretion from the E.coli genome-wide mutant library.The precise coupling of protein localization with sucrose sensitivity is achieved by fusing the target protein to the levansucrase from Bacillus subtilis(SacB).Ultimately,we isolated two key factors.DnaK and CRP,through this screening system.As a molecular chaperone,DnaK may play an important role in the transmembrane transport of BcsZ,and the global transcriptional regulator CRP may play a specific regulatory role in this process.In conclusion,our research has deepened the understanding of BcsZ transmembrane translocation in E.coli and laid the foundation for its secretion mechanism.