[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-382] Amino acid deaminase outperforms transaminase in α-keto acid production from amino acids

○Ryo Nasuno¹, Hisashi Kudo¹, Keiji Fushimi², Ryota Hidese¹, Akihiko Kondo^1,2,3, Tomohisa Hasunuma^1,2,3 (1. Engineering Biology Research Center, Kobe University (Japan), 2. Graduate School of Science, Technology, and Innovation, Kobe University (Japan), 3. Research Center for Sustainable Resource Science, RIKEN (Japan))

Keywords:

Deaminase,Transaminase,α-Keto acid,α-Amino acid,Microbial production

α-Keto acids are central metabolic intermediates that serve as precursors for a broad range of valuable compounds, including alcohols, organic acids, and alkanes. Therefore, efficient biosynthesis of α-keto acids from α-amino acids is essential for microbial production of α-keto acids-derived products. While transaminases have traditionally been employed for this conversion, their dependence on α-ketoglutarate and reversibility can impose metabolic burdens and limit yield. Recently, amino acid deaminases (AADs) have been used for conversion of amino acids to α-keto acids, because AADs catalyze irreversible oxidative deamination without any co-substrate and thus may offer advantages for α-keto acid production. Here, we experimentally demonstrated the superiority of AADs over transaminases in amino acid-to-α-keto acid conversion for the first time, using the production of indole-3-acetic acid from tryptophan as a case study. Escherichia coli cells expressing AAD genes from Proteus myxofaciens or P. mirabilis exhibited higher production of indole-3-acetic acid and indole-3-pyruvic acid, which is a direct product from tryptophan and an intermediate for indole-3-acetic acid production, than transaminases. Our kinetic analysis revealed that AADs exhibited higher affinity towards tryptophan than transaminases, contributing to higher production rate. This is the first report to demonstrate the superiority of AADs over transaminases for α-keto acids production. Furthermore, AADs showed better performance in the bioconversion of leucine, valine, and phenylalanine to their corresponding α-keto acids than transaminases. These findings establish AADs as promising biocatalysts for α-keto acid synthesis, supporting their use in pathway design for bioproduction of α-keto acid-derived compounds.We further investigated the substrate specificities of AADs derived from Proteus myxofaciens (PmyxAAD) and Proteus vulgaris (PvAAD). PmyxAAD showed high activity especially to aromatic amino acids, while PvAAD exhibited the broad substrate specificity with higher activity to all 22 amino acids tested. Notably, a single amino acid substitution near the predicted substrate-binding pocket (Gln278Leu) altered the substrate preference of PvAAD, enhancing its specificity toward aromatic amino acids. These findings provide mechanistic insights into substrate recognition in AADs and offer a basis for rational enzyme engineering.