Rational molecular engineering of l-amino acid deaminase for production of α-ketoisovaleric acid from l-valine by Escherichia coli

Abstract

The targeted modification of enzymatic efficiency can drive an increased production of desired metabolites. α-Ketoisovaleric acid (KIV) is a candidate material for use in the pharmaceutical and food industries. In the present study, we aimed to enhance the biotransformation efficiency of L-amino acid deaminase (L-aad) from Proteus myxofaciens ATCC 19692 to improve the production of KIV from L-valine. First, L-aad was expressed in Escherichia coli BL21(DE3). We employed transformed E. coli cells as a whole-cell biocatalyst system and optimized their biochemical characteristics for the biotransformation of L-valine. Then, based on the known 3D structural model of L-aad from P. myxofaciens and the simulation results for docking with L-valine, four amino acid residues (N100, Q276, R316, and F318) were identified as potential target sites for mutagenesis. Next, we performed site-directed saturation mutagenesis to improve the biotransformation efficiency. With 11.3 g L⁻¹ L-valine, the bioconversion efficiencies of a single-mutant strain (F318T) and a double-mutant strain (F318T and N100H) were 4.474 and 8.197 g L⁻¹, respectively, whereas that of the wild-type strain was 2.014 g L⁻¹ under optimal conditions. In summary, we developed a one-step process for KIV production via expressing P. myxofaciens L-aad in E. coli BL21(DE3) and enhanced the yield of KIV by site-directed saturation mutagenesis of L-aad.