Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.
We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.
Galkin
A
A
Institute for Chemical Research, Kyoto University, Japan.
Kulakova
L
L
Yoshimura
T
T
Soda
K
K
Esaki
N
N
D050788:Alanine Dehydrogenase; D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism; D000410:Alanine Transaminase / Q000235:genetics / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D051263:D-Alanine Transaminase; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D007651:Keto Acids / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
D000596:Amino Acids; D017931:DNA Primers; D007651:Keto Acids; D011994:Recombinant Proteins; D005560:Formate Dehydrogenases; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase; D000411:Alanine Racemase