Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve alpha-keto acid production-from the corresponding l-amino acids-catalyzed by FAD-dependent l-amino acid deaminase (l-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis. In the resulting E. coli strain PHCF7, strong overexpression of pma, ribC, and ribF and moderate overexpression of ribH yielded a 90% increase in phenylpyruvic acid (PPA) titer: 19.4 +/- 1.1 g.L-1. Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH(2)/FAD using FDH for FADH(2)/FAD regeneration and NOX for NAD(+)/NADH regeneration. The resulting E. coli strain PHCF7-FDH-NOX yielded the highest PPA production: 31.4 +/- 1.1 g.L-1. Finally, this whole-cell system was adapted to production of other alpha-keto acids including alpha-ketoglutaric acid, alpha-ketoisocaproate, and keto-gamma-methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH(2)/FAD regeneration for production of alpha-keto acids. Notably, the strategy reported herein may be generally applicable to other flavin-dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. (C) 2017 Wiley Periodicals, Inc.