Concept of N-Acetylglucosamine Fermentation
N-acetylglucosamine (NAG) was identified as a more favorable fermentation product than glucosamine. It was stable at a large range of pH. It did not inhibit cell growth when incubated at higher concentrations in the medium. NAG could also be easily hydrolyzed to glucosamine under relatively mild acidic conditions. Therefore, different options of metabolic engineering for NAG production in E. coli were evaluated.
Significant amounts of NAG were produced by over-expressing E. coli glmM (phosphoglucosamine mutase) and glmU (bifunctional enzyme glucosamine-1-P N-acetyltransferase and N-acetylglucosamine-1-P uridyltransferase, see Figure 1). However, the over-expressed endogenous pathway was relatively inefficient for NAG production. NAG titer was only a few grams per liter and more glucosamine was detected in the growth medium.
Unlike bacteria, yeast, plants and animals have an enzyme called GNA1, which converts glucosamine-6-P into N-acetylglucosamine-6-P. Over-expression of a GNA1 gene in a glucosamine production strain led to the synthesis and secretion of high levels of NAG in the growth medium (Figure 7). The acetylation step was so efficient that very little glucosamine was produced as a by-product (1 g/L).
Figure 7. Engineered Metabolic Pathway for N-Acetylglucosamine Production in E. coli