Engineering leucine dehydrogenase to enhance L-2-aminobutyric acid production in Escherichia coli
摘要
L-2-aminobutyric acid (L-ABA) is a non-proteinogenic chiral α-amino acid and, as an important chemical feedstock and pharmaceutical intermediate, has broad application potential in the pharmaceutical and fine chemical industries. In this study, Escherichia coli TWF106 was used as the chassis strain for L-ABA production. In strain TWF106, we individually deleted rhtA, rhtC, ptsG and gabP to optimize the metabolic flux toward L-ABA production, and constructed multiple single-deletion and double-deletion strains. The reductive amination of 2-ketobutyric acid (2-KB) to L-ABA catalyzed by leucine dehydrogenase (LeuDH) is considered a potential bottleneck in this pathway; therefore, we engineered LeuDH to improve the L-ABA titer. By substituting the key residue Ser331 in LeuDH with different amino acids, 19 mutants were constructed and tested for L-ABA production. Two mutants, LeuDHS331R and LeuDHS331C, showed the greatest improvement in L-ABA production, the L-ABA titers of TWF106-331R and TWF106-331 C reached 5.46 g/L and 5.45 g/L respectively. Molecular docking and molecular dynamics simulations revealed that mutations at Ser331 altered the static binding mode and modulated the dynamic binding mechanism of the protein–ligand complex. Subsequently, by combining metabolic flux optimization with site-directed mutagenesis, TWF1604-331 C was constructed through deletion of gabP in TWF106 coupled with introduction of plasmid pB-AmDHS331C. TWF1604-331 C could produce 6.47 g/L L-ABA. Finally, fed-batch fermentation of TWF1604-331 C was conducted in a 2-L bioreactor, achieving an L-ABA titer of 16.58 g/L.