Enhanced Conversion Rate of L-Phenylalanine by Coupling Reactions of Aminotransferases and Phosphoenolpyruvate Carboxykinase in Escherichia coli K-12

Abstract

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.

Asymmetric synthesis of L‐homophenylalanine by equilibrium‐shift using recombinant aromatic L‐amino acid transaminase

“…A high concentration of ␣-keto acid usually inhibits ␣-transaminase reaction as either a substrate or a product (Chao et al, 1999). To screen the microorganisms having transaminase activity for the asymmetric synthesis of L-HPA with relaxed substrate inhibition by 2-OPBA, its reverse reaction was used with the minimal media containing 50 mM L-HPA and 100 mM glycerol as a sole nitrogen and carbon source (Gokul et al, 2000;Shin and Kim, 2001), respectively.…”

“…2) Asymmetric transamination can also be used to convert corresponding achiral keto acid substrates into the required stereoisomers without any extra steps, as in kinetic resolution reactions (Ager et al, 1997;Fotheringham et al, 1999;Stewart, 2001). Aspartate transaminase coupled with phosphoenolpyruvate carboxykinase was used to produce Lphenylalanine from phenylpyruvate with 93% conversion yield (Chao et al, 1999). Here, the phosphoenolpyruvate carboxykinase was incorporated to convert the byproduct oxaloacetate into phosphoenolpyruvate more efficiently in the recombinant E. coli cell.…”

“…Although many attempts at the asymmetric synthesis of amino acids were reported using the transaminase, most of them suffered from low equilibrium constant of the reaction and substrate and product inhibitions by keto acids in aqueous buffer solution, and these problems became the critical disadvantage of using the transaminase (Chao, et al, 1999). In an in vitro system, the substrate inhibition can be easily reduced by using a fed-batch type of reactor operation.…”

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

“…Transaminases (TAs) have been studied extensively due to their use in the production of amino acids and chiral amines (5,18,19). TAs play an important role in amino acid metabolism and are ubiquitous in both microorganisms and eukaryotic cells (6,18).…”

“…Despite their advantages, including broad substrate specificity and high stability (19), the industrial use of these enzymes has been limited due to the low equilibrium constants of the TA reactions. However, by removing one of the products via coupling to other enzymes or whole-cell biotransformation, D and L amino acids have been successfully produced (3,5,17,19).…”