Efficient Synthesis of D-Phenylalanine from L-Phenylalanine via a Tri-Enzymatic Cascade Pathway

D-phenylalanine is an important intermediate in food and pharmaceutical industries. Here, to enable efficient D-phenylalanine biosynthesis from L-phenylalanine, a tri-enzymatic cascade was designed and reconstructed in vivo. The activity of Proteus vulgaris meso-diaminopimelate dehydrogenase (PvDAPDH) toward phenyl pyruvic acid was identified as the limiting step. To overcome, the tension in the phenyl pyruvic acid side-chain, PvDAPDH was engineered, generating PvDAPDH(W121A/R181S/H227I), whose catalytic activity of 6.86 U mg(-1) represented an 85-fold increase over PvDAPDH. Introduction of PvDAPDH(W121A/R181S/H227I), P. mirabilis L-amino acid deaminase, and Bacillus megaterium glucose dehydrogenase in E. coli enabled the production of 57.8 g L-1 D-phenylalanine in 30 h, the highest titer to date using 60 g L-1 L-phenylalanine as starting substrate, which meant a 96.3 % conversion rate and >99 % enantioselectivity on a 3-L scale. The proposed tri-enzymatic cascade provides a novel potential bio-based approach for industrial production of D-phenylalanine from cheap amino acids.

Automated summary

The study successfully achieved efficient production of D-phenylalanine from L-phenylalanine through the engineering of a tri-enzymatic cascade system, providing a new bio-based approach for industrial production of D-phenylalanine from cheap amino acids.