Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α-keto acids.

Abstract

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 α-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 FADH2 /FAD using FDH for FADH2 /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 α-keto acids including α-ketoglutaric acid, α-ketoisocaproate, and keto-γ-methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH2 /FAD regeneration for production of α-keto acids. Notably, the strategy reported herein may be generally applicable to other flavin-dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. 2017;114: 1928-1936. © 2017 Wiley Periodicals, Inc.