Development of a potential stationary-phase specific gene expression system by engineering of SigB-dependent cg3141 promoter in Corynebacterium glutamicum

Abstract Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promot...
Ausführliche Beschreibung

Abstract Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promoters is desired, which can be activated without expensive inducers and has significant advantages for industrial-scale use. Here, we developed a stationary-phase gene expression system by engineering a sigma factor B (SigB)-dependent promoter that can be activated during the transition phase between exponential and stationary growth phases in C. glutamicum. First, the inducibilities of three well-known SigB-dependent promoters were examined using super-folder green fluorescent protein as a reporter protein, and we found that promoter of cg3141 ($ P_{cg3141} $) exhibited the highest inducibility. Next, a synthetic promoter library was constructed by randomizing the flanking and space regions of $ P_{cg3141} $, and the stationary-phase promoters exhibiting high strengths were isolated via FACS-based high-throughput screening. The isolated synthetic promoter ($ P_{4-N14} $) showed a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the use of the isolated $ P_{4-N14} $ for fed-batch cultivation was verified with the production of glutathione S-transferase as a model protein in a lab-scale (5-L) bioreactor. Ausführliche Beschreibung