Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis.
Appl Microbiol Biotechnol
; 108(1): 184, 2024 Jan 30.
Article
em En
| MEDLINE
| ID: mdl-38289384
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Escherichia coli
/
Aminobutiratos
/
Transaminases
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article