Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium Glutamicum .

Background: Corynebacterium glutamicum ( C. glutamicum ) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins.

Objectives: In this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum .

Materials and Methods: The synthesized cloning/expression cassette C was assembled into the basic E. coli - C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum /pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein.

Results: The vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5- amyE . The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5- amyE , not in the cell lysate of 14067/pAU5- amyE . The α-amylase specific activity of the culture supernatant of 14067/pAU5- amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5- amyE . These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum /pAU5 system.

Conclusions: A novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum . The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/ pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.