Typical N-acetyl-d-neuraminic acid (Neu5Ac) production uses N-acetyl-d-glucosamine (GlcNAc) and excess pyruvate as substrates in the enzymatic or whole-cell biocatalysis process. In a previous study, a Neu5Ac-producing biocatalytic process via engineered Escherichia coli SA-05/pDTrc-AB/pCDF-pck-ppsA was constructed without exogenous pyruvate. In this study, glycerol was found to be a good energy source compared with glucose for the catalytic system with resting cells, and Neu5Ac production increased to 13.97 ± 0.27 g L−1. In addition, a two-stage pH shift strategy was carried out, and the Neu5Ac yield was improved to 14.61 ± 0.31 g L−1. The GlcNAc concentration for Neu5Ac production was optimized. Finally, an integrated strategy was developed for Neu5Ac production, and the Neu5Ac yield reached as high as 18.17 ± 0.27 g L−1. These results provide a new biocatalysis technology for Neu5Ac production without exogenous pyruvate.