Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis

Abstract

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM‐1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. Key points • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization.

One-Pot Two-Stage Biocatalytic Cascade to Produce l-Phosphinothricin by Two Enantioselective Complementary Aminotransferases at High Substrate Loading via a Deracemization Process

“…15−18 To date, several natural (S)selective ATs have been reported for the synthesis of L-PPT from its prochiral ketone, exhibiting satisfactory catalytic activity and strong tolerance to high concentrations of substrate. 9,19,20 However, the use of (R)-selective ATs for the kinetic resolution of D and L-PPT has rarely been reported in previous studies. Adaption to special ecological environments often leads to the evolution of enzymes with extraordinary properties.…”

“…As an alternative approach, the deracemization of unnatural amino acids using enantiocomplementary aminotransferases as catalysts has considerable potential for achieving stringent stereoselectivity with no redox cofactors required. Aminotransferase (AT)-based deracemization processes have been employed in the synthesis of various unnatural chiral amino acids such as l -homophenylalanine and ( S )-2-aminobutyrate, which are important chiral intermediates in pharmaceutical drugs. − To date, several natural ( S )-selective ATs have been reported for the synthesis of L-PPT from its prochiral ketone, exhibiting satisfactory catalytic activity and strong tolerance to high concentrations of substrate. ,, However, the use of ( R )-selective ATs for the kinetic resolution of D and L-PPT has rarely been reported in previous studies.…”