Development and characterization of expression vectors for Corynebacterium glutamicum.

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including Ptac, Psod, Psod with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, PilvC, PilvC with a conserved SD-1 (PilvC-M1), and PilvC with a conserved SD-2 (PilvC-M2), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, Psod and Psod-M were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and Psod-M displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of PilvC > PilvC-M1 > Psod > PilvC-M2 > Psod-M, whereas all plasmids except pCSP30 with Psod displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with Psod-M, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.