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Efficient Synthesis of D‐Phenylalanine from L‐Phenylalanine via a Tri‐Enzymatic Cascade Pathway
Author(s) -
Lu Cui,
Zhang Sheng,
Song Wei,
Liu Jia,
Chen Xiulai,
Liu Liming,
Wu Jing
Publication year - 2021
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.202100237
Subject(s) - phenylalanine , chemistry , enzyme , corynebacterium glutamicum , amino acid , biosynthesis , biochemistry , stereochemistry , dehydrogenase , gene
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.