Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis

Glucosamine (GlcN) and its acetylated derivative N-acetylglucosamine (GlcNAc) are widely used in the pharmaceutical industries. Here, we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum. Specifically, we ligated the GNA1 gene, which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate, into the plasmid pJYW4 and then transformed this recombinant vector into the C. glutamicum ATCC 13032, ATCC 13869, ATCC 14067, and S9114 strains, and we assessed the GlcNAc titers at 0.5 g/L, 1.2 g/L, 0.8 g/L, and 3.1 g/L from each strain, respectively. This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C. glutamicum strains. Therefore, we performed whole genome sequencing of the S9114 strain, which has not been previously published, and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested. Next, nagA (encoding GlcNAc-6-phosphate deacetylase) and gamA (encoding GlcN-6-phosphate deaminase) were deleted in C. glutamicum S9114 to block the catabolism of intracellular GlcNAc, leading to a 54.8% increase in GlcNAc production (from 3.1 to 4.8 g/L) when grown in a shaker flask. In addition, lactate synthesis was blocked by knockout of ldh (encoding lactate dehydrogenase); thus, further increasing the GlcNAc titer to 5.4 g/L. Finally, we added a key gene of the GlcN synthetic pathway, glmS, from different sources into the expression vector pJYW-4-ceN, and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L. This is the first report concerning the metabolic engineering of C. glutamicum, and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.