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 . Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH /FAD using FDH for FADH /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 . 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 FADH /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.

Production of riboflavin and related cofactors by biotechnological processes

“…As an important cofactor, FAD plays a key role in many enzymatic reactions. An increased intracellular FAD concentration in E. coli improved the production of phenylpyruvic acid (PPA) [144]. Genes related to the synthesis of FAD, including ribADBHCF, were overexpressed individually to investigate their effects on the intracellular FAD concentration, and the results suggested that reactions catalyzed by ribC (encoding RF synthase), ribF (encoding the bifunctional RFK/FADS) and ribH (encoding lumazine synthase) were the main rate-limiting steps for FAD biosynthesis.…”

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

“…Lee, Park, & Kim, ), in which the overexpression of an oxygen‐carrying protein (bacterial hemoglobin) in an E. coli strain improved the transformation of cyclohexanone into caprolactone. On the other hand, the FAD content of E. coli is roughly estimated to a few µmoles per g CDW (Hou et al, ), namely the same order of magnitude as the production of BVMO (Figure a). As suggested by Milker, Goncalves, Fink, and Rudroff, (), the endogenous FAD maybe not enough to form the active FAD‐bound BVMO, necessary to the reaction.…”

“…Regarding the enzyme ratio, another limiting factor probably interfered to explain the plateau observed Figure 4c, possibly the NADPH/NADP + content, O 2 supply, and/or the endogenous FAD content. NADPH limitation is well documented On the other hand, the FAD content of E. coli is roughly estimated to a few µmoles per g CDW (Hou et al, 2017), namely the same order of magnitude as the production of BVMO (Figure 4a). As suggested by Milker, Goncalves, Fink, and Rudroff, (2017), the endogenous FAD maybe not enough to form the active FADbound BVMO, necessary to the reaction.…”

A new approach for efficient synthesis of phenyllactic acid from L-phenylalanine: Pathway design and cofactor engineering

“…Compared to phenylalanine dehydrogenase (Brunhuber, Thoden, Blanchard, & Vanhooke, ; Ödman, Wellborn, & Bommarius, ) and aminotransferases (Hirotsu, Goto, Okamoto, & Miyahara, ), the oxidative deamination reaction l ‐AAD catalyzed requires only one amino acid, and can synthesize PPA without extra addition of coenzyme (FAD) or coenzyme regeneration system, because the FAD E.coli itself produced can support the entire reaction, so l ‐AAD was the optimal one. In addition, Hou et al has obtained the triple mutant D165K/F263M/L336M of l ‐AAD, which produced the highest PPA titer of 10.0 ± 0.4 g·L −1 of resting‐cell biotransformation, with a substrate conversion ratio of 100% (Hou et al, ; Hou, Hossain, Li, Shin, Du, et al, ; Hou, Hossain, Li, Shin, Liu, et al, ). Therefore, the triple mutant of l‐aad was cloned first to generate plasmid pRSF‐aad in this study.…”

“…In previous work, we found that l ‐amino acid deaminases ( l ‐AAD) from Proteus mirabilis KCTC 2566 can catalyze the efficient deamination of l ‐phenylalanine (Phe) to PPA and ammonia with a substrate conversion ratio of 100% (Hou et al, ; Hou, Hossain, Li, Shin, Du, et al, ; Hou, Hossain, Li, Shin, Liu, et al, ). The price of Phe is much lower than that of PPA (the price of PPA is about 500 times that of Phe), and thus the production cost of PLA can be significantly decreased if with Phe, not PPA, is used as the substrate.…”