Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase.
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.
Cho
Byung-Kwan
BK
Interdisciplinary Program for Biochemical Engineering and Biotechnology, School of Chemical Engineering, and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
Seo
Joo-Hyun
JH
Kang
Tae-Won
TW
Kim
Byung-Gee
BG
D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004754:Enterobacter / Q000201:enzymology; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000613:Aminobutyrates; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; C014328:2-amino-4-phenylbutyric acid; D000637:Transaminases; C019729:aromatic amino acid aminotransferase