Enhanced production of N -acetyl- d -neuraminic acid by whole-cell bio-catalysis of Escherichia coli

N-acetyl- d -neuraminic acid (Neu5Ac) has been considerably focused due to its promising potential applications in pharmaceuticals and dairy products. A whole-cell biocatalyst process is an important tool for synthesis of pharmaceutical intermediates and fine chemicals. In this study, a whole cell process using engineered Escherichia coli strain was developed and stepwise optimization was employed for Neu5Ac production. N-acetyl- D -glucosamine 2-epimerase and Neu5Ac aldolase were overexpressed in E. coli individually and the activity ratio was optimized by varying recombinant amounts of cell biomass for synthesis of Neu5Ac. Moreover, substrate concentrations and ratio of pyruvate and N-acetyl- D -glucosamine (GlcNAc) and detergent concentrations were optimized to increase product synthesis. The resulting process generated 237.4 mM Neu5Ac with a yield of 40.0% mol/mol GlcNAc. Furthermore, transporter pathways involved in Neu5Ac and GlcNAc were engineered and their impact on the Neu5Ac synthesis was evaluated. Using a stepwise optimization, an overall whole-cell biocatalytic process was developed and a maximum titer of 260.0 mM Neu5Ac (80.4 g/L) with a conversion yield of 43.3% from GlcNAc was achieved. The process can be used for industrial large-scale production of Neu5Ac in terms of efficiency and economy.