Metabolic engineering of cofactor FLAVIN ADENINE DINUCLEOTIDE (cas 146-14-5) (FAD) synthesis and regeneration in Escherichia coli for production of α‐keto acids

Add time:09/28/2019    Source:infona.pl

Cofactor FLAVIN ADENINE DINUCLEOTIDE (cas 146-14-5) (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.

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