Abstract D-phenylglycine is an industrially important non-proteinogenic amino acid, which so far can only be produced by classical or chemoenzymatic synthesis. Based on the new identified L-phenylglycine biosynthetic pathway from the pristinamycin producer Streptomyces pristinaespiralis and a stereoinverting aminotransferase gene ( hpgAT ) from Pseudomonas putida , an artificial D-phenylglycine operon was constructed, which can be used for the fermentative production of D-phenylglycine.