Cascade biocatalysis for enantioselective reconstruction of both enantiomers of phenylalaninol from biobased l-phenylalanine

Abstract

Enantiopure phenylalaninols are vital in pharmaceuticals and bioactive compounds. Catalytic carboxylic acid reduction is a promising way to synthesize these compounds from biobased L-phenylalanine. However, traditional methods face issues like harsh reaction conditions, costly metal catalysts, significant waste production, and safety concerns. To address these issues, we developed a one-pot cascade biocatalysis system that converts biobased L-phenylalanine into both enantiomers of phenylalaninol through sequential deamination, decarboxylation, hydroxymethylation, and asymmetric reductive amination. We identified a novel benzaldehyde lyase (RpBAL) from Rhodopseudomonas palustris, which shows broad substrate tolerance for aryl aliphatic aldehydes and benzaldehydes, achieving conversion rates of 20%–99% for producing α-hydroxy ketones. Homology modelling, molecular docking, and molecular dynamics simulations were performed to elucidate the highly efficient catalytic mechanisms of RpBAL for phenylacetaldehyde. By using engineered recombinant Escherichia coli (EAL-RR) co-expressing L-amino acid deaminase (LAAD), α-keto acid decarboxylase (ARO10), and RpBAL, along with E. coli (ATA) cells expressing amine transaminase, we efficiently converted biobased L-phenylalanine into (R)- and (S)-phenylalaninol in a one-pot two-stage reaction, achieving conversions of 72% and 80% with >99% ee. This biocatalytic approach was successfully applied to synthesize (R)- and (S)-phenylalaninol on a 150 mg scale, obtaining 60–70% isolated product yields with >99.0% ee. Notably, (R)-phenylalaninol can be directly modified in one step to produce solriamfetol, an approved drug for treating excessive daytime sleepiness. This study presents a novel, green, and sustainable approach for synthesizing enantiopure phenylalaninol from biobased L-phenylalanine, effectively overcoming the limitations of traditional chemical methods and showcasing the potential of biocatalytic cascade processes in pharmaceutical manufacturing.