L-Dihydroxyphenylalanine (DOPA) was biosynthesized by a tyrosine-phenol lyase from catechol, pyruvate, and ammonia in Escherichia coli, and the biosynthesized amino acid was directly incorporated into proteins. Three biochemical experiments with mutant proteins containing DOPA confirmed the genetic incorporation of biosynthesized DOPA, and revealed its potential for various biochemical applications.

Four unnatural amino acids (UAAs) were synthesized from their corresponding α-keto acids by using glutamine:phenylpyruvate aminotransferase (GlnAT) from Thermus thermophilus HB8. The enzyme efficiently catalyzed the conversion of the unnatural substrates, with little decrease in its activity compared to that shown toward its natural substrates. The α-keto acids were converted into the corresponding amino acids in cells and then directly incorporated into a protein with high efficiency and fidelity. Because α-keto acids have no chiral center and are synthetically readily accessible, this method will further enhance the genetic incorporation technology and may present a new approach to the preparation of optically pure UAAs. Considering the substrate promiscuity of the GlnAT observed in the study, this method can be applied to the genetic incorporation of other UAAs derived from Tyr or Phe, which constitute approximately 50% of the successfully incorporated UAAs. In addition, this study provides an impetus for the development of an autonomous unnatural organism that can synthesize and incorporate UAAs.