A Tri‐Enzyme Cascade for Efficient Production of L‐2‐Aminobutyrate from L‐Threonine

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 ( Bt LDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism‐based protein engineering generated the optimal triple variant Bt LDH M3 (A262S/V296C/P150M) with 20.7‐fold increased specific activity and 9.6‐fold increased k cat / K m compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 : 1:0.6) of these enzymes in vivo close to the optimal ratio (0.4 : 1 : 1) in vitro . Importing the optimal triple mutant Bt LDH M3 into our constructed pathway in vivo and optimization of transformation conditions achieved one‐pot conversion of L‐Thr to 130.2 g/L L‐ABA, with 95 % conversion, 99 % e.e . and 10.9 g L −1 h −1 productivity (the highest to date) in 12 h on a 500 mL scale. These results describe a potential biosynthesis approach for the industrial production of L‐ABA.