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

Abstract

L-Homophenylalanine (L-HPA) was asymmetrically synthesized from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two-step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K-1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314-fold higher than that of the wild-type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2-OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L-HPA at high concentrations of 2-OPBA. To avoid the substrate inhibition by 2-OPBA, intermittent addition of the solid-state substrate was attempted to obtain a high concentration of L-HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L-HPA at 840 mM of 2-OPBA resulted in >94% of conversion yield and >99% ee of L-HPA of optical purity. Due to the low solubility (<2 mM) of L-HPA in the reaction buffer, synthesized L-HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L-HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction.

Cloning and Characterization of a Novel β-Transaminase fromMesorhizobiumsp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids

“…Moreover, transaminases allow asymmetric synthesis from prochiral ketone compounds depending upon the properties of target chemical compounds (1,4,10,11,17,49,50). Though transaminases are not widespread, we have reported an -transaminase of Alcaligenes denitrificans which can catalyze mainly the transamination between aliphatic ␤-amino acids and pyruvate (56).…”

Engineering aromaticL-amino acid transaminase for the asymmetric synthesis of constrained analogs ofL-phenylalanine

“…Chiral resolution processes were disclosed in a myriad of reports using pronase, lipase, transaminase, and hydantoinase (Houng et al, 1996;Pugniere et al, 1994;Shin and Kim, 1997;Syldatk et al, 1992). Finally, asymmetric transamination could also be used to convert achiral keto acid substrates into the stereoisomers without any additional steps (Ager et al, 1997;Alexeeva et al, 2002;Cho et al, 2003;Fotheringham et al, 1999;Stewart, 2001;Stirling et al, 1992). However, it has not been reported yet that the constrained analogs of L-phenylalanine such as L-diphenylalanine were synthesized via enzymatic methods.…”

“…The pET24ma (constructed by Dr. David Sourdive, Pasteur Institute, France) containing p15A replication origin was used to construct the plasmids carrying AroATEs gene (Cho et al, 2003). The structural gene of AroATEs was amplified from the genomic DNA of Enterobacter sp.…”

“…BK2K-1 showed unique substrate specificity toward aromatic amino acids, such as L-phenylalanine, L-tyrosine, and L-homophenylalanine (Cho et al, 2003). However, AroATEs has the limited applications in synthesizing the constrained analogs of L-phenylalanine such as L-diphenylalanine.…”

“…To address this need, we have paid attention to the transaminase technology for the large-scale biosynthesis of enantiomerically pure unnatural amino acids via asymmetric synthesis due to its broad substrate specificity and no requirement of external cofactor regeneration (Cho et al, 2003;Fotheringham et al, 1997). Transaminases are versatile pyridoxal-5 0 -phosphate (PLP)-dependent enzymes, which execute reversible transamination reactions between amino acids and keto acids in amino acid metabolism (Christen and Metzler, 1985).…”