One-step biosynthesis of α-keto-γ- Methylthiobutyric acid from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered lamino acid deaminase from proteus vulgaris

Abstract

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of L-methionine, has great potential for use as an alternative to L-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase (L-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris L-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of L-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L L-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40 °C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris L-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.