Large-scale engineering of the Corynebacterium glutamicum genome

Abstract

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.

FIG. 1.

Illustration of SSIs larger than 10 kb in the C. glutamicum R genome. The lengths are the following: for 1, 15.6 kb; for 2, 11.1 kb; for 3, 56.2 kb; for 4, 22.6 kb; for 5, 32.8 kb; for 6, 16.2 kb; for 7, 16.5 kb; for 8, 45.1 kb; for 9, 16.3 kb; for 10, 14.6 kb; and for 11, 12.0 kb.

FIG. 2.

Schematic representation of Cre/loxP-mediated deletion of the C. glutamicum R genome. Km^r, Sp^r, and cat indicate kanamycin, spectinomycin, and chloramphenicol resistance genes, respectively. GR1 and 2 are short segments of the C. glutamicum R genome. These segments were amplified by PCR and integrated into plasmids for homologous recombination. Black arrows (P1 and P2) under the chromosome represent PCR primers.

FIG. 3.

The result of Cre expression in order to delete the SSIs. The upper and lower parts of each plate are from after and before transformation of pCRA406. The numbers on the plate indicate the SSI used for deletion experiments. Cells were plated on complex medium containing (A) kanamycin and spectinomycin or (B) no antibiotics and incubated for 24 h at 33°C. (C) Verification of SSI deletion was conducted by PCR. Direct cell PCR was performed, and about 2-kb DNA fragments were successfully amplified with P1 and P2 primers with the Cre⁺ strain. No fragments were observed from the strains before transformation of pCRA406. M indicates the marker.