A Tri-Enzyme Cascade for Efficient Production of L-2-Aminobutyrate from L-Threonine.
Chembiochem
; 24(15): e202300148, 2023 08 01.
Article
em En
| MEDLINE
| ID: mdl-36946691
ABSTRACT
L-2-aminobutyrate (L-ABA) is an important chiral drug intermediate with a key role in modern medicinal chemistry. Here, we describe the development of an efficient method for the asymmetric synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using a cost-effective L-Thr. Low activity of leucine dehydrogenase from Bacillus thuringiensis (BtLDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism-based protein engineering generated the optimal triple variant BtLDHM3 (A262S/V296C/P150M) with 20.7-fold increased specific activity and 9.6-fold increased kcat /Km compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 10.6) of these enzymes inâ
vivo close to the optimal ratio (0.4 1 1) inâ
vitro. Importing the optimal triple mutant BtLDHM3 into our constructed pathway inâ
vivo and optimization of transformation conditions achieved one-pot conversion of L-Thr to 130.2â
g/L L-ABA, with 95 % conversion, 99 % e.e. and 10.9â
g L-1 h-1 productivity (the highest to date) in 12â
h on a 500â
mL scale. These results describe a potential biosynthesis approach for the industrial production of L-ABA.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Treonina
/
Escherichia coli
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article