A novel genetic tool for metabolic optimization of Corynebacterium glutamicum: efficient and repetitive chromosomal integration of synthetic promoter-driven expression libraries.

Fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. We have developed a tool for this purpose for the important industrial workhorse Corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. We first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pJS31 serving as the carrier. The pJS31 vector contains a synthetic cassette including a phage attachment site attP for integration, a bacterial attachment site attB for subsequent integration, a multiple cloning site, and two modified loxP sites to facilitate easy removal of undesirable vector elements. Meanwhile, we constructed a derivative of the wild-type strain ATCC 13032 carrying an attB site in its chromosome (JS34) and demonstrated that pJS31 readily could integrate into the attB site in this strain providing expression of the corresponding integrase. Subsequent expression of the Cre recombinase promoted recombination between the modified loxP sites, resulting in a strain only retaining the target insertions and an attB site. To simplify the procedure, non-replicating circular expression units for the phage integrase and the Cre recombinase were used. As a showcase, we used the tool to construct a battery of strains simultaneously expressing the two reporter genes, lacZ (encoding β-galactosidase) and gusA (encoding β-glucuronidase), to arbitrary levels. In principle, an unlimited number of genes, whether native, heterologous, or synthetic, can be introduced using the developed approach, and this should greatly facilitate metabolic optimization of this important platform organism.