Development of an Escherichia coli-based biocatalytic system for the efficient synthesis of N-acetyl-D-neuraminic acid.

N-acetyl-d-neuraminic acid (Neu5Ac) is a valuable resource that has seen increasing demand in both medicine and biotechnology. Although enzymatic systems and whole-cell biocatalysts have been developed for the synthesis of Neu5Ac, low yield and productivity still hamper the use of these methods on larger scales. We report the creation of an Escherichia coli biocatalyst for the efficient synthesis of Neu5Ac using a metabolic and protein engineering strategy. Expression of the two enzymes, N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac lyase (NAL), was balanced using promoter engineering. Genes encoding competing pathways and GlcNAc catabolism were deleted, and then a structure-guided process was used to identify a more efficient NAL and an AGE mutant with a higher rate of Neu5Ac synthesis. The resulting biocatalyst produced 351.8 mM Neu5Ac with a yield of 58.6% from GlcNAc. This work exemplifies the use of rational design and protein engineering to construct a complex bacterial biocatalyst that can serve as a platform for the large-scale synthesis of a useful biological material.