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

Graphical We developed a pathway for the synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using L-Thr, and the expression of three enzymes was optimized by vector design. We solved the crystal structure of the key enzyme LDH and proposed protein engineering strategies based on the structure to improve the catalytic activity of the enzyme. 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.