Construction of an expression vector that uses the aph promoter for protein expression in Corynebacterium glutamicum

Corynebacterium glutamicum is an attractive host for the production of heterologous proteins despite its traditional use in fermentative production of amino acids. To enhance the expression levels of target genes, the development of useful promoters is required in the construction of expression systems. Here, we developed a new promoter, the aph promoter from aminoglycoside-3'-phosphotransferase gene, and used it to construct monocistronic and bicistronic expression systems that host different ribosome binding site (RBS) sequences. First, the expression level of the reporter protein, enhanced green fluorescent protein (EGFP), varied with changes in the RBS sequences in the constructed vectors. The results showed that the fluorescence intensities of the bicistronic group were higher than those of the monocistronic group and that RM3E showed the highest fluorescence intensity, which was 42-fold higher than the lowest (RA2E') among these groups. Next, taking advantage of the optimized aph promoter, we successfully employed this aph promoter for α-amylase and V_HH (camelid antibody fragment) expression. The secretion of α-amylase improved 1.5-fold after promoter mutation. This promoter will be useful for heterologous protein production in C. glutamicum cells.