Construction of a novel expression system for use in Corynebacterium glutamicum

• Developed a novel expression system for C. glutamicum.
• Constructed plasmids pJYW-4 and pJYW-6 for the expression system.
• Deleted the aceE gene in three different C. glutamicum strains by using the system.
• Deleted both aceE and ilvA genes in C. glutamicum ATCC14067 by using the system.

Corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. Suitable vectors are needed for metabolic engineering in C. glutamicum. Most available vectors used in C. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. These vectors, though excellent for laboratory use, are not preferable choices for industry-scale fermentation. In this work, we developed a novel expression system for use in C. glutamicum, which do not require antibiotics when used for industrial fermentation. This system includes two vectors: the shuttle vector pJYW-4 for expression of genes and the vector pJYW-6 for deletion of the essential gene alr in C. glutamicum. The vector pJYW-4 contains a large multiple cloning site for cloning multiple genes and two selective markers: one is the kanamycin-resistant gene kan and the other is an essential gene alr. The selective marker kan facilitates molecular manipulation or fermentations in the laboratory, and the selection marker alr is good for use in industry-scale fermentation, allowing in vivo maintenance of the expression vector through auxotrophic complementation; therefore, the two selection markers in pJYW-4 make it useful for both laboratory research and industrial fermentation, and convenient to transfer valuable laboratory-developed strains into industrial production. This newly-constructed expression system was successfully used to increase l-valine production in C. glutamicum ATCC 14067, indicating its potential on developing amino acid-producing C. glutamicum strains.